Opioid receptor antagonist prodrugs

ABSTRACT

Provided herein are prodrugs of opioid receptor antagonists such as nalmefene and naltrexone, pharmaceutical compositions comprising said compounds, and methods for using said compounds for the treatment of behavioral disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of InternationalApplication No. PCT/CN2018/113850, filed on Nov. 3, 2018, which claimsbenefit of U.S. Provisional Patent Application No. 62/581,501 filed onNov. 3, 2017, and U.S. Provisional Patent Application No. 62/697,289filed on Jul. 12, 2018. The entire contents of the aforementionedapplication are incorporated herein by reference.

BACKGROUND

A need exists in the medicinal arts for compositions and methods for themodulation of opioid receptor activity in the course of treatingbehavioral disorders,

BRIEF SUMMARY OF THE INVENTION

Provided herein are prodrugs of opioid receptor antagonists such asnalmefene and naltrexone, pharmaceutical compositions comprising saidcompounds, and methods for using said compounds for the treatment ofbehavioral disorders.

Some compounds of the invention have superior properties. For example,some compounds of the invention have superior stabilities in oil basedpharmaceutical compositions such as sesame oil or cottonseed oil.

Some compounds of the invention have better pharmacokinetic activitiesin vivo (for example, rat or dog), e.g., extended half-life.

Some compounds of the invention have better safety in vivo (for example,rat or dog), e.g., diminished injection site reactions.

Some compounds of the invention have superior stability either neat orin oil based pharmaceutical compositions.

In one aspect, provided herein is a compound, or pharmaceuticallyacceptable salt thereof, having a structure provided in Formula (I),

wherein,

X is O or CH₂;

R is selected from:

a. (C₃-C₇cycloalkyl)CH₂C(O)—;

b. (C₃-C₇cycloalkyl)CH₂CH₂C(O)—;

c. —C(O)OC₇-C₂₀ alkyl; or

d. —C(O)NHC(CH₃)₃.

In some embodiments, X is O. In some embodiments, X is CH₂.

In another aspect, also provided herein is a compound, orpharmaceutically acceptable salt thereof, having a structure provided inFormula (II),

wherein,

X is O or CH₂;

R is:

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀alkenyl; and n is 7-15; providedif X is O, then n is not 7.

In some embodiments, X is O. In some embodiments, X is CH₂.

In one aspect, provided herein is a compound, or pharmaceuticallyacceptable salt thereof, having a structure provided in Formula (II),

wherein,

X is O or CH₂;

R is

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl; and n is 7-15;provided if X is O, then n is not 7.

In some embodiments, X is O. In some embodiments, X is CH₂.

In another aspect, also provided herein is a compound, orpharmaceutically acceptable salt thereof, having a structure provided inFormula (IIa),

wherein,

X is O or CH₂;

R is:

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl; and n is 9-15.

In some embodiments, X is O. In some embodiments, X is CH₂.

In another aspect, also provided herein is a compound, orpharmaceutically acceptable salt thereof, having a structure provided inFormula (III),

wherein,

X is O or CH₂;

R is selected from:

-   -   —[CH(R³)O]z-R⁴;    -   —[CH(R³)O]z-C(═O)OR⁴;    -   —[CH(R³)O]z-C(═O)NR⁴R⁵; and    -   —[CH(R³)O]z-P(═O)(OR⁴)(OR⁵);

wherein z is 1, 2, 3, 4, 5, 6, or 7;

R³ is hydrogen, halogen, alkyl, alkenyl, cycloalkylalkyl, or aryl;

each R⁴ and R⁵ is independently selected from hydrogen, alkyl, alkenyl,cycloalkylalkyl, or aryl.

In some embodiments, X is O. In some embodiments, X is CH₂.

One embodiment provides a pharmaceutical composition comprising apharmaceutically acceptable excipient and a compound of any one ofFormula (I), (II), (IIa), or (III), or a compound disclosed in Table 1,or a pharmaceutically acceptable salt thereof.

Provided herein is a method of treating opioid dependence in a patientin need thereof comprising administering a pharmaceutical compositioncomprising a compound of Formula (I), (II), (IIa), or (III), or acompound disclosed in Table 1, or pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable excipient.

One embodiment provides a method of treating a patient wherein thetherapeutic effect of a long acting opioid antagonist depot can beovercome in a patient by administering an opioid based analgesic.

One embodiment provides a method of treating opioid dependence in apatient in need thereof, wherein the patient receives a first injectionof an injectable formulation comprising a compound of any one of Formula(I), (II), (IIa), or (III), or a compound disclosed in Table 1, or apharmaceutically acceptable salt thereof, wherein said first injectionprovides a therapeutically relevant plasma concentration for about 1week, about 2 weeks, about 3 weeks or about 4 weeks, at least about 2months, at least about 3 months, at least about 4 months, at least about5 months or at least about 6 months, followed by a second injection ofan injectable formulation comprising a compound of any one of Formula(I), (II), (IIa), or (III), or a compound disclosed in Table 1, or apharmaceutically acceptable salt thereof, wherein said second injectionprovides a therapeutically relevant plasma concentration for at leastabout 2 months, at least about 3 months, at least about 4 months, atleast about 5 months or at least about 6 months.

One embodiment provides a method of treating opioid dependence in apatient in need thereof, wherein the patient receives a first injectionof an injectable formulation of naltrexone loaded PLGA microspheres thatprovides a therapeutically relevant plasma concentration for about 4weeks, followed by one or more injections of an injectable formulationcomprising a compound of any one of Formula (I), (II), (IIa), or (III),or a compound disclosed in Table 1, or a pharmaceutically acceptablesalt thereof, that provides a therapeutically relevant plasmaconcentration for about 2 months, about 3 months, about 4 months, orabout 5 months or more.

One embodiment provides a method of treating opioid dependence in apatient in need thereof, wherein the patient receives one or moreinjections of an injectable formulation comprising at least one compoundof any one of Formula (I), (II), (IIa), or (III), or a compounddisclosed in Table 1, or a pharmaceutically acceptable salt thereof,wherein the patient has been previously treated with opioid agonists orpartial agonists, such as buprenorphine or methadone, and the patientsare now transitioning to discontinuation from such agonist or partialagonist treatment.

One embodiment provides a method of treating opioid dependence in apatient in need thereof, wherein the patient receives one or moreinjections of an injectable formulation comprising at least one compoundof any one of Formula (I), (II), (IIa), or (III), or a compounddisclosed in Table 1, or a pharmaceutically acceptable salt thereof,wherein the patient is recently addicted and nave to prior medicationassisted treatment, or wherein the patient has recently discontinuedopioid pain medication, are at risk of future opioid drug abuse, and arein need of prevention of future opioid drug abuse via antagonisttreatment.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference for the specificpurposes identified herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings below. The patent application file contains atleast one drawing executed in color. Copies of this patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIG. 1 provides the nuclear magnetic resonance spectrum of Example 1(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl((E)-octadec-9-en-1-yl) carbonate;

FIG. 2 provides the nuclear magnetic resonance spectrum of Example 2(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylundecyl carbonate;

FIG. 3 provides the nuclear magnetic resonance spectrum of Example 3(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylundecyl carbonate;

FIG. 4 provides the nuclear magnetic resonance spectrum of Example 4(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylundecyl carbonate; and

FIG. 5 provides the nuclear magnetic resonance spectrum of Example 5(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylundecyl carbonate.

FIG. 6 provides the nuclear magnetic resonance spectrum of Example 6(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldodecyl carbonate;

FIG. 7 provides the nuclear magnetic resonance spectrum of Example 7(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl((E)-octadec-9-en-1-yl) carbonate;

FIG. 8 provides the nuclear magnetic resonance spectrum of Example 8(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(E)-octadec-9-enoate;

FIG. 9 provides the nuclear magnetic resonance spectrum of Example 9(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl(E)-octadec-9-enoate;

FIG. 10 provides the nuclear magnetic resonance spectrum of Example 10(4aS,7aR,12bS:(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9decylcarbonate;

FIG. 11 provides the nuclear magnetic resonance spectrum of Example 11(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldodecyl carbonate;

FIG. 12 provides the nuclear magnetic resonance spectrum of Example 12(4aS,7as,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylstearate;

FIG. 13 provides the nuclear magnetic resonance spectrum of Example 13(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(Z)-docos-13-enoate;

FIG. 14 provides the nuclear magnetic resonance spectrum of Example 14(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldocosanoate;

FIG. 15 provides the nuclear magnetic resonance spectrum of Example 15(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(E)-octadec-9-enoate;

FIG. 16 provides the nuclear magnetic resonance spectrum of Example 16(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylicosanoate;

FIG. 17 provides the nuclear magnetic resonance spectrum of Example 17(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yloctyl carbonate;

FIG. 18 provides the nuclear magnetic resonance spectrum of Example 18(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldecyl carbonate;

FIG. 19 provides the nuclear magnetic resonance spectrum of Example 19(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylhexadecyl carbonate;

FIG. 20 provides the nuclear magnetic resonance spectrum of Example 20(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(9Z,12Z,15Z)-octadeca-9,12,15-trienoate;

FIG. 21 provides the nuclear magnetic resonance spectrum of Example 21of(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylhexadecyl carbonate;

FIG. 22 provides the nuclear magnetic resonance spectrum of Example 22(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(Z)-docos-13-enoate;

FIG. 23 provides the nuclear magnetic resonance spectrum of Example 23(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yloctyl carbonate;

FIG. 24 provides the nuclear magnetic resonance spectrum of Example 24(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyldodecyl carbonate;

FIG. 25 provides the nuclear magnetic resonance spectrum of Example 25(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyltetradecyl carbonate;

FIG. 26 provides the nuclear magnetic resonance spectrum of Example 26(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl(E)-octadec-9-enoate;

FIG. 27 provides the nuclear magnetic resonance spectrum of Example 27(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyltetradecyl carbonate;

FIG. 28 provides the nuclear magnetic resonance spectrum of Example 28(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylicosyl carbonate;

FIG. 29 provides the nuclear magnetic resonance spectrum of Example 29(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyldodecyl carbonate;

FIG. 30 provides the nuclear magnetic resonance spectrum of Example 30(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yltridecyl carbonate;

FIG. 31 provides the nuclear magnetic resonance spectrum of Example 31(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yltetradecyl carbonate;

FIG. 32 provides the nuclear magnetic resonance spectrum of Example 32(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylpentadecyl carbonate;

FIG. 33 provides the nuclear magnetic resonance spectrum of Example 33(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yloctadecyl carbonate;

FIG. 34 provides the nuclear magnetic resonance spectrum of Example 34(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylhexadecyl carbonate;

FIG. 35 provides the nuclear magnetic resonance spectrum of Example 35(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyldecyl carbonate;

FIG. 36 provides the nuclear magnetic resonance spectrum of Example 36(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yloleate;

FIG. 37 provides the nuclear magnetic resonance spectrum of Example 37(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(9Z,12Z)-octadeca-9,12-dienoate;

FIG. 38 provides the nuclear magnetic resonance spectrum of Example 38(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl3,3-dimethylbutanoate;

FIG. 39 provides the nuclear magnetic resonance spectrum of Example 39(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl3-cyclopentylpropanoate;

FIG. 40 provides the nuclear magnetic resonance spectrum of Example 40(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yltert-butylcarbamate;

FIG. 41 provides the nuclear magnetic resonance spectrum of Example 41(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yloleate;

FIG. 42 provides the nuclear magnetic resonance spectrum of Example 42(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl3,3-dimethylbutanoate;

FIG. 43 provides the nuclear magnetic resonance spectrum of Example 43(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl3-cyclopentylpropanoate;

FIG. 44 provides the nuclear magnetic resonance spectrum of Example 44(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyldodecanoate;

FIG. 45 provides the nuclear magnetic resonance spectrum of Example 45(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyltetradecanoate;

FIG. 46 provides the nuclear magnetic resonance spectrum of Example 46(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylhexadecanoate.

FIG. 47 provides the nuclear magnetic resonance spectrum of Example 47(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylhexadecyl carbonate;

FIG. 48 provides the nuclear magnetic resonance spectrum of Example 48(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyldodecanoate; and

FIG. 49 provides the nuclear magnetic resonance spectrum of Example 49(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylhexadecanoate.

DETAILED DESCRIPTION OF THE INVENTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an agent” includesa plurality of such agents, and reference to “the cell” includesreference to one or more cells (or to a plurality of cells) andequivalents thereof known to those skilled in the art, and so forth.When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. The term “about” when referring toa number or a numerical range means that the number or numerical rangereferred to is an approximation within experimental variability (orwithin statistical experimental error), and thus the number or numericalrange. In some instances, will vary between 1% and 15% of the statednumber or numerical range. The term “comprising” (and related terms suchas “comprise” or “comprises” or “having” or “including”) is not intendedto exclude that in other certain embodiments, for example, an embodimentof any composition of matter, composition, method, or process, or thelike, described herein, “consist of” or “consist essentially of” thedescribed features.

Definitions

As used in the specification and appended claims, unless specified tothe contrary, the following terms have the meaning indicated below.

“Amino” refers to the —NH₂ radical.

“Cyano” refers to the —CN radical.

“Nitro” refers to the —NO₂ radical.

“Oxa” refers to the —O— radical.

“Oxo” refers to the ═O radical.

“Thioxo” refers to the ═S radical.

“Imino” refers to the ═N—H radical.

“Oximo” refers to the ═N—OH radical.

“Hydrazino” refers to the ═N—NH₂ radical.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to fifteen carbon atoms (e.g., C₁-C₁₅alkyl). In certain embodiments, an alkyl comprises one to thirteencarbon atoms (e.g., C₁-C₁₃ alkyl). In certain embodiments, an alkylcomprises one to eight carbon atoms (e.g., C₁-C₈ alkyl). In otherembodiments, an alkyl comprises one to five carbon atoms (e.g., C₁-C₅alkyl). In other embodiments, an alkyl comprises one to four carbonatoms (e.g., C₁-C₄ alkyl). In other embodiments, an alkyl comprises oneto three carbon atoms (e.g., C₁-C₃ alkyl). In other embodiments, analkyl comprises one to two carbon atoms (e.g., C₁-C₂ alkyl). In otherembodiments, an alkyl comprises one carbon atom (e.g., C₁ alkyl). Inother embodiments, an alkyl comprises five to fifteen carbon atoms(e.g., C₅-C₁₅ alkyl). In other embodiments, an alkyl comprises five toeight carbon atoms (e.g., C₅-C₈ alkyl). In other embodiments, an alkylcomprises two to five carbon atoms (e.g., C₂-C₅ alkyl). In otherembodiments, an alkyl comprises three to five carbon atoms e.g., C₃-C₅alkyl). In other embodiments, the alkyl group is selected from methyl,ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl(n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl),1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl isattached to the rest of the molecule by a single bond. Unless statedotherwise specifically in the specification, an alkyl group isoptionally substituted by one or more of the following substituents:halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —OC(O)—N(R^(a))₂,—N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1 or 2)and —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) isindependently hydrogen, alkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), carbocyclylalkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl),heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), heterocyclylalkyl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl), orheteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy,or trifluoromethyl).

“Alkoxy” refers to a radical bonded through an oxygen atom of theformula —O-alkyl, where alkyl is an alkyl chain as defined above.

“Alkenyl” refers to a straight or branched hydrocarbon chain radicalgroup consisting solely of carbon and hydrogen atoms, containing atleast one carbon-carbon double bond, and having from two to twelvecarbon atoms. In certain embodiments, an alkenyl comprises two to eightcarbon atoms. In other embodiments, an alkenyl comprises two to fourcarbon atoms. The alkenyl is attached to the rest of the molecule by asingle bond, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e.,allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like. Unlessstated otherwise specifically in the specification, an alkenyl group isoptionally substituted by one or more of the following substituents:halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl,—OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a),—C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a), —OC(O)—N(R^(a))₂,—N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2),—S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is 1 or 2)and —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) isindependently hydrogen, alkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), carbocyclylalkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl),heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), heterocyclylalkyl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl), orheteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy,or trifluoromethyl).

“Alkynyl” refers to a straight or branched hydrocarbon chain radicalgroup consisting solely of carbon and hydrogen atoms, containing atleast one carbon-carbon triple bond, having from two to twelve carbonatoms. In certain embodiments, an alkynyl comprises two to eight carbonatoms. In other embodiments, an alkynyl comprises two to six carbonatoms. In other embodiments, an alkenyl comprises two to four carbonatoms. The alkenyl is attached to the rest of the molecule by a singlebond, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, andthe like. Unless stated otherwise specifically in the specification, analkynyl group is optionally substituted by one or more of the followingsubstituents: halo, cyano, nitro, oxo, thioxo, imino, oximo,trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a), —N(R^(a))₂,—C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂, —N(R^(a))C(O)OR^(a),—OC(O)—N(R^(a))₂, —N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where t is1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2), —S(O)_(t)R^(a) (where t is1 or 2) and —S(O)_(t)N(R^(a))₂ (where t is 1 or 2) where each R^(a) isindependently hydrogen, alkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), carbocyclylalkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl),heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), heterocyclylalkyl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl), orheteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy,or trifluoromethyl).

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, containing no unsaturation andhaving from one to twelve carbon atoms, for example, methylene,ethylene, propylene, n-butylene, and the like. The alkylene chain isattached to the rest of the molecule through a single bond and to theradical group through a single bond. The points of attachment of thealkylene chain to the rest of the molecule and to the radical group isthrough one carbon in the alkylene chain or through any two carbonswithin the chain. In certain embodiments, an alkylene comprises one toeight carbon atoms (e.g., C₁-C₈ alkylene). In other embodiments, analkylene comprises one to five carbon atoms (e.g., C₁-C₅ alkylene). Inother embodiments, an alkylene comprises one to four carbon atoms (e.g.,C₁-C₄ alkylene). In other embodiments, an alkylene comprises one tothree carbon atoms (e.g., C₁-C₃ alkylene). In other embodiments, analkylene comprises one to two carbon atoms (e.g., C₁-C₂ alkylene). Inother embodiments, an alkylene comprises one carbon atom (e.g.,alkylene). In other embodiments, an alkylene comprises five to eightcarbon atoms (e.g., C₅-C₈ alkylene). In other embodiments, an alkylenecomprises two to five carbon atoms (e.g., C₂-C₅ alkylene). In otherembodiments, an alkylene comprises three to five carbon atoms (e.g.,C₃-C₅ alkylene). Unless stated otherwise specifically in thespecification, an alkylene chain is optionally substituted by one ormore of the following substituents: halo, cyano, nitro, oxo, thioxo,imino, oximo, trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a),—N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —OC(O)—N(R^(a))₂, —N(R^(a))C(O)R^(a),—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)R^(a) (where t is 1 or 2) and —S(O)_(t)N(R^(a))₂(where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), aryl (optionally substituted with halogen, hydroxy,methoxy, or trifluoromethyl), aralkyl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl),heterocyclylalkyl (optionally substituted with halogen, hydroxy,methoxy, or trifluoromethyl), heteroaryl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl).

“Alkynylene” or “alkynylene chain” refers to a straight or brancheddivalent hydrocarbon chain linking the rest of the molecule to a radicalgroup, consisting solely of carbon and hydrogen, containing at least onecarbon-carbon triple bond, and having from two to twelve carbon atoms.The alkynylene chain is attached to the rest of the molecule through asingle bond and to the radical group through a single bond. In certainembodiments, an alkynylene comprises two to eight carbon atoms (e.g.,C₂-C₈ alkynylene). In other embodiments, an alkynylene comprises two tofive carbon atoms (e.g., C₂-C₅ alkynylene). In other embodiments, analkynylene comprises two to four carbon atoms (e.g., C₂-C₄ alkynylene).In other embodiments, an alkynylene comprises two to three carbon atoms(e.g., C₂-C₃ alkynylene). In other embodiments, an alkynylene comprisestwo carbon atom (e.g., C₂ alkylene). In other embodiments, an alkynylenecomprises five to eight carbon atoms (e.g., C₅-C₈ alkynylene). In otherembodiments, an alkynylene comprises three to five carbon atoms (e.g.,C₃-C₅ alkynylene). Unless stated otherwise specifically in thespecification, an alkynylene chain is optionally substituted by one ormore of the following substituents: halo, cyano, nitro, oxo, thioxo,imino, oximo, trimethylsilanyl, —OR^(a), —SR^(a), —OC(O)—R^(a),—N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —OC(O)—N(R^(a))₂, —N(R^(a))C(O)R^(a),—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —S(O)_(t)OR^(a) (where t is1 or 2), —S(O)_(t)R^(a) (where t is 1 or 2) and —S(O)_(t)N(R^(a))₂(where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), aryl (optionally substituted with halogen, hydroxy,methoxy, or trifluoromethyl), aralkyl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl),heterocyclyl alkyl (optionally substituted with halogen, hydroxy,methoxy, or trifluoromethyl), heteroaryl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl).

“Aryl” refers to a radical derived from an aromatic monocyclic ormulticyclic hydrocarbon ring system by removing a hydrogen atom from aring carbon atom. The aromatic monocyclic or multicyclic hydrocarbonring system contains only hydrogen and carbon from five to eighteencarbon atoms, where at least one of the rings in the ring system isfully unsaturated, i.e., it contains a cyclic, delocalized (4n+2)π-electron system in accordance with the Hückel theory. The ring systemfrom which aryl groups are derived include, but are not limited to,groups such as benzene, fluorene, indane, indene, tetralin andnaphthalene. Unless stated otherwise specifically in the specification,the term “aryl” or the prefix “ar-” (such as in “aralkyl”) is meant toinclude aryl radicals optionally substituted by one or more substituentsindependently selected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl,cyano, nitro, optionally substituted aryl, optionally substitutedaralkyl, optionally substituted aralkynyl, optionally substitutedaralkynyl, optionally substituted carbocyclyl, optionally substitutedcarbocyclylalkyl, optionally substituted heterocyclyl, optionallysubstituted heterocyclylalkyl, optionally substituted heteroaryl,optionally substituted heteroarylalkyl, —R^(b)—OR^(a),—R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR^(a), —R^(b)—OC(O)—N(R^(a))₂,—R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a),—R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a),—R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a)(where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2) and—R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), where each R^(a) isindependently hydrogen, alkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), cycloalkylalkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl),heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), heterocyclylalkyl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl), orheteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy,or trifluoromethyl), each R^(b) is independently a direct bond or astraight or branched alkylene or alkenylene chain, and R^(c) is astraight or branched alkylene or alkenylene chain, and where each of theabove substituents is unsubstituted unless otherwise indicated.

“Aralkyl” refers to a radical of the formula —R^(c)-aryl where R^(c) isan alkylene chain as defined above, for example, methylene, ethylene,and the like. The alkylene chain part of the aralkyl radical isoptionally substituted as described above for an alkylene chain. Thearyl part of the aralkyl radical is optionally substituted as describedabove for an aryl group.

“Aralkenyl” refers to a radical of the formula —R^(d)-aryl where R^(d)is an alkenylene chain as defined above. The aryl part of the aralkenylradical is optionally substituted as described above for an aryl group.The alkenylene chain part of the aralkenyl radical is optionallysubstituted as defined above for an alkenylene group.

“Aralkynyl” refers to a radical of the formula —R^(e)-aryl, where R^(e)is an alkynylene chain as defined above. The aryl part of the aralkynylradical is optionally substituted as described above for an aryl group.The alkynylene chain part of the aralkynyl radical is optionallysubstituted as defined above for an alkynylene chain.

“Aralkoxy” refers to a radical bonded through an oxygen atom of theformula —O—R^(c)-aryl where R^(c) is an alkylene chain as defined above,for example, methylene, ethylene, and the like. The alkylene chain partof the aralkyl radical is optionally substituted as described above foran alkylene chain. The aryl part of the aralkyl radical is optionallysubstituted as described above for an aryl group.

“Carbocyclyl” refers to a stable non-aromatic monocyclic or polycyclichydrocarbon radical consisting solely of carbon and hydrogen atoms,which includes fused or bridged ring systems, having from three tofifteen carbon atoms. In certain embodiments, a carbocyclyl comprisesthree to ten carbon atoms. In other embodiments, a carbocyclyl comprisesfive to seven carbon atoms. The carbocyclyl is attached to the rest ofthe molecule by a single bond. Carbocyclyl is saturated (i.e.,containing single C—C bonds only) or unsaturated (i.e., containing oneor more double bonds or triple bonds). A fully saturated carbocyclylradical is also referred to as “cycloalkyl.” Examples of monocycliccycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl. An unsaturated carbocyclyl isalso referred to as “cycloalkenyl.” Examples of monocyclic cycloalkenylsinclude, e.g., cyclopentenyl, cyclohexenyl, cycloheptenyl, andcyclooctenyl Polycyclic carbocyclyl radicals include, for example,adamantyl, norbornyl (i.e., bicyclo[2,2,1]heptanyl), norbornenyl,decalinyl, 7,7-dimethyl-bicyclo[2,2.1]heptanyl, and the like. Unlessotherwise stated specifically in the specification, the term“carbocyclyl” is meant to include carbocyclyl radicals that areoptionally substituted by one or more substituents independentlyselected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo,cyano, nitro, optionally substituted aryl, optionally substitutedaralkyl, optionally substituted aralkenyl, optionally substitutedaralkynyl, optionally substituted carbocyclyl, optionally substitutedcarbocyclylalkyl, optionally substituted heterocyclyl, optionallysubstituted heterocyclylalkyl, optionally substituted heteroaryl,optionally substituted heteroarylalkyl, R^(b)—OR^(a),—R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—N(R^(a))₂,—R^(b)—N(R^(a))₂, —R^(b)C(O)R^(a), —R^(b)—C(O)OR^(a),—R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a),—R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a)(where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2) and—R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), where each R^(a) isindependently hydrogen, alkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), cycloalkylalkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl),heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), heterocyclylalkyl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl), orheteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy,or trifluoromethyl), each R^(b) is independently a direct bond or astraight or branched alkylene or alkenylene chain, and R^(c) is astraight or branched alkylene or alkenylene chain, and where each of theabove substituents is unsubstituted unless otherwise indicated.

“Carbocyclylalkyl” refers to a radical of the formula —R^(c)-carbocyclylwhere R^(c) is an alkylene chain as defined above. The alkylene chainand the carbocyclyl radical is optionally substituted as defined above.

“Carbocyclylalkynyl” refers to a radical of the formula—R^(c)-carbocyclyl where R^(c) is an alkynylene chain as defined above.The alkynylene chain and the carbocyclyl radical is optionallysubstituted as defined above.

“Carbocyclylalkoxy” refers to a radical bonded through an oxygen atom ofthe formula —O—R^(c)-carbocyclyl where R^(c) is an alkylene chain asdefined above. The alkylene chain and the carbocyclyl radical isoptionally substituted as defined above.

As used herein, “carboxylic acid bioisostere” refers to a functionalgroup or moiety that exhibits similar physical, biological and/orchemical properties as a carboxylic acid moiety. Examples of carboxylicacid bioisosteres include, but are not limited to,

and the like.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodosubstituents.

“Fluoroalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more fluoro radicals, as defined above, forexample, trifluoromethyl, difluoromethyl, fluoromethyl,2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, and the like. Insome embodiments, the alkyl part of the fluoroalkyl radical isoptionally substituted as defined above for an alkyl group.

“Heterocyclyl” refers to a stable 3- to 18-membered non-aromatic ringradical that comprises two to twelve carbon atoms and from one to sixheteroatoms selected from nitrogen, oxygen and sulfur. Unless statedotherwise specifically in the specification, the heterocyclyl radical isa monocyclic, bicyclic, tricyclic or tetracyclic ring system, whichoptionally includes fused or bridged ring systems. The heteroatoms inthe heterocyclyl radical are optionally oxidized. One or more nitrogenatoms, if present, are optionally quaternized. The heterocyclyl radicalis partially or fully saturated. The heterocyclyl is attached to therest of the molecule through any atom of the ring(s). Examples of suchheterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl,octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl,2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl,4-piperidonyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl,tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl,thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.Unless stated otherwise specifically in the specification, the term“heterocyclyl” is meant to include heterocyclyl radicals as definedabove that are optionally substituted by one or more substituentsselected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo,cyano, nitro, optionally substituted aryl, optionally substitutedaralkyl, optionally substituted aralkenyl, optionally substitutedaralkynyl, optionally substituted carbocyclyl, optionally substitutedcarbocyclylalkyl, optionally substituted heterocyclyl, optionallysubstituted heterocyclylalkyl, optionally substituted heteroaryl,optionally substituted heteroarylalkyl, —R^(b)—OR^(a),—R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR^(a), —R^(b)—OC(O)—N(R^(a))₂,—R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a), —R^(b)—C(O)OR^(a),—R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a),—(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a) (where tis 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2) and—R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), where each R^(a) isindependently hydrogen, alkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), cycloalkylalkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl),heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), heterocyclylalkyl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl), orheteroarylalkyl. (optionally substituted with halogen, hydroxy, methoxy,or trifluoromethyl), each R^(b) is independently a direct bond or astraight or branched alkylene or alkenylene chain, and R^(c) is astraight or branched alkylene or alkenylene chain, and where each of theabove substituents is unsubstituted unless otherwise indicated.

“N-heterocyclyl” or “N-attached heterocyclyl” refers to a heterocyclylradical as defined above containing at least one nitrogen and where thepoint of attachment of the heterocyclyl radical to the rest of themolecule is through a nitrogen atom in the heterocyclyl radical. AnN-heterocyclyl radical is optionally substituted as described above forheterocyclyl radicals. Examples of such N-heterocyclyl radicals include,but are not limited to, 1-morpholinyl, 1-piperidinyl, 1-piperazinyl,1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl.

“C-heterocyclyl” or “C-attached heterocyclyl” refers to a heterocyclylradical as defined above containing at least one heteroatom and wherethe point of attachment of the heterocyclyl radical to the rest of themolecule is through a carbon atom in the heterocyclyl radical. AC-heterocyclyl radical is optionally substituted as described above forheterocyclyl radicals. Examples of such C-heterocyclyl radicals include,but are not limited to, 2-morpholinyl, 2- or 3- or 4-piperidinyl,2-piperazinyl, 2- or 3-pyrrolidinyl, and the like.

“Heterocyclylalkyl” refers to a radical of the formula—R^(c)-heterocyclyl where R^(c) is an alkylene chain as defined above.If the heterocyclyl is a nitrogen-containing heterocyclyl, theheterocyclyl is optionally attached to the alkyl radical at the nitrogenatom. The alkylene chain of the heterocyclylalkyl radical is optionallysubstituted as defined above for an alkylene chain. The heterocyclylpart of the heterocyclylalkyl radical is optionally substituted asdefined above for a heterocyclyl group.

“Heterocyclylalkoxy” refers to a radical bonded through an oxygen atomof the formula —O—R^(c)-heterocyclyl where R^(c) is an alkylene chain asdefined above. If the heterocyclyl is a nitrogen-containingheterocyclyl, the heterocyclyl is optionally attached to the alkylradical at the nitrogen atom. The alkylene chain of theheterocyclylalkoxy radical is optionally substituted as defined abovefor an alkylene chain. The heterocyclyl part of the heterocyclylalkoxyradical is optionally substituted as defined above for a heterocyclylgroup.

“Heteroaryl” refers to a radical derived from a 3- to 18-memberedaromatic ring radical that comprises two to seventeen carbon atoms andfrom one to six heteroatoms selected from nitrogen, oxygen and sulfur.As used herein, the heteroaryl radical is a monocyclic, bicyclic,tricyclic or tetracyclic ring system, wherein at least one of the ringsin the ring system is fully unsaturated, i.e., it contains a cyclic,delocalized (4n+2) π-electron system in accordance with the Hückeltheory. Heteroaryl includes fused or bridged ring systems. Theheteroatom(s) in the heteroaryl radical is optionally oxidized. One ormore nitrogen atoms, if present, are optionally quaternized. Theheteroaryl is attached to the rest of the molecule through any atom ofthe ring(s). Examples of heteroaryls include, but are not limited to,azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl,benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl,benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl,benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,cyclopenta[d]pyrimidinyl,6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl,5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl,6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl,dibenzothiophenyl, furanyl, furanonyl, furo[3,2-c]pyridinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl,indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl,isoquinolyl, indolizinyl, isoxazolyl,5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl,5,6,6a,7,8,9,10,1.0a-octahydrobenzo[h]quinazolinyl,1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl,phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl,pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl,quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl,tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl,5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl,5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl,triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl,thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl (i.e.thienyl). Unless stated otherwise specifically in the specification, theterm “heteroaryl” is meant to include heteroaryl radicals as definedabove which are optionally substituted by one or more substituentsselected from alkyl, alkenyl, alkynyl, halo, fluoroalkyl, haloalkenyl,haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl,optionally substituted aralkyl, optionally substituted aralkenyl,optionally substituted aralkynyl, optionally substituted carbocyclyl,optionally substituted carbocyclylalkyl, optionally substitutedheterocyclyl, optionally substituted heterocyclylalkyl, optionallysubstituted heteroaryl, optionally substituted heteroarylalkyl,—R^(b)—OR^(a), —R^(b)—OC(O)—R^(a), —R^(b)—OC(O)—OR^(a),—R^(b)—OC(O)—N(R^(a))₂, —R^(b)—N(R^(a))₂, —R^(b)—C(O)R^(a),—R^(b)—C(O)OR^(a), —R^(b)—C(O)N(R^(a))₂, —R^(b)—O—R^(c)—C(O)N(R^(a))₂,—R^(b)—N(R^(a))C(O)OR^(a), —R^(b)—N(R^(a))C(O)R^(a),—R^(b)—N(R^(a))S(O)_(t)R^(a) (where t is 1 or 2), —R^(b)—S(O)_(t)R^(a)(where t is 1 or 2), —R^(b)—S(O)_(t)OR^(a) (where t is 1 or 2) and—R^(b)—S(O)_(t)N(R^(a))₂ (where t is 1 or 2), where each R^(a) isindependently hydrogen, alkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl(optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), cycloalkylalkyl (optionally substituted with halogen,hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl),heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, ortrifluoromethyl), heterocyclylalkyl (optionally substituted withhalogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionallysubstituted with halogen, hydroxy, methoxy, or trifluoromethyl), orheteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy,or trifluoromethyl), each R^(b) is independently a direct bond or astraight or branched alkylene or alkenylene chain, and R^(c) is astraight or branched alkylene or alkenylene chain, and where each of theabove substituents is unsubstituted unless otherwise indicated.

“N-heteroaryl” refers to a heteroaryl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heteroaryl radical to the rest of the molecule is through a nitrogenatom in the heteroaryl radical. An N-heteroaryl radical is optionallysubstituted as described above for heteroaryl radicals.

“C-heteroaryl” refers to a heteroaryl radical as defined above and wherethe point of attachment of the heteroaryl radical to the rest of themolecule is through a carbon atom in the heteroaryl radical. AC-heteroaryl radical is optionally substituted as described above forheteroaryl radicals.

“Heteroarylalkyl” refers to a radical of the formula —R^(c)-heteroaryl,where R^(c) is an alkylene chain as defined above. If the heteroaryl isa nitrogen-containing heteroaryl, the heteroaryl is optionally attachedto the alkyl radical at the nitrogen atom. The alkylene chain of theheteroarylalkyl radical is optionally substituted as defined above foran alkylene chain. The heteroaryl part of the heteroarylalkyl radical isoptionally substituted as defined above for a heteroaryl group.

“Heteroarylalkoxy” refers to a radical bonded through an oxygen atom ofthe formula —O—R^(c)-heteroaryl, where R^(c) is an alkylene chain asdefined above. If the heteroaryl is a nitrogen-containing heteroaryl,the heteroaryl is optionally attached to the alkyl radical at thenitrogen atom. The alkylene chain of the heteroarylalkoxy radical isoptionally substituted as defined above for an alkylene chain. Theheteroaryl part of the heteroarylalkoxy radical is optionallysubstituted as defined above for a heteroaryl group.

The compounds disclosed herein. In some embodiments, contain one or moreasymmetric centers and thus give rise to enantiomers, diastereomers, andother stereoisomeric forms that are defined, in terms of absolutestereochemistry, as (R)— or (S)—. Unless stated otherwise, it isintended that all stereoisomeric forms of the compounds disclosed hereinare contemplated by this disclosure. When the compounds described hereincontain alkene double bonds, and unless specified otherwise, it isintended that this disclosure includes both E and Z geometric isomers(e.g., cis or trans.) Likewise, all possible isomers, as well as theirracemic and optically pure forms, and all tautomeric forms are alsointended to be included. The term “geometric isomer” refers to E or Zgeometric isomers (e.g., cis or trans of an alkene double bond. The term“positional isomer” refers to structural isomers around a central ring,such as ortho-, meta-, and para-isomers around a benzene ring.

A “tautomer” refers to a molecule wherein a proton shift from one atomof a molecule to another atom of the same molecule is possible. Thecompounds presented herein, in certain embodiments, exist as tautomers.In circumstances where tautomerization is possible, a chemicalequilibrium of the tautomers will exist. The exact ratio of thetautomers depends on several factors, including physical state,temperature, solvent, and pH. Some examples of tautomeric equilibriuminclude:

The compounds disclosed herein. In some embodiments, are used indifferent enriched isotopic forms, e.g., enriched in the content of ²H,³H, ¹¹C, ¹³C and/or ¹⁴C. In one particular embodiment, the compound isdeuterated in at least one position. Such deuterated forms can be madeby the procedure described in U.S. Pat. Nos. 5,846,514 and 6,334,997. Asdescribed in U.S. Pat. Nos. 5,846,514 and 6,334,997, deuteration canimprove the metabolic stability and or efficacy, thus increasing theduration of action of drugs.

Unless otherwise stated, structures depicted herein are intended toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of the present disclosure.

The compounds of the present disclosure optionally contain unnaturalproportions of atomic isotopes at one or more atoms that constitute suchcompounds. For example, the compounds may be labeled with isotopes, suchas for example, deuterium (²H), tritium (³H), iodine-125 (¹²⁵I) orcarbon-14 (¹⁴C). Isotopic substitution with 2H, ¹¹C, ¹³C, ¹⁴C, ¹⁵C, ¹²N,¹³N, ¹⁵N, ¹⁶N, ¹⁶O, ¹⁷O, ¹⁴F, ¹⁵F, ¹⁶F, ¹⁷F, ¹⁸F, ³³S, ³⁴S, ³⁵S, ³⁶S,³⁵O, ³⁷O, ⁷⁹Br, ⁸¹Br, ¹²⁵I are all contemplated. All isotopic variationsof the compounds of the present invention, whether radioactive or not,are encompassed within the scope of the present invention.

In certain embodiments, the compounds disclosed herein have some or allof the ¹H atoms replaced with ²H atoms. The methods of synthesis fordeuterium-containing compounds are known in the art and include, by wayof non-limiting example only, the following synthetic methods.

Deuterium substituted compounds are synthesized using various methodssuch as described in: Dean, Dennis C.; Editor. Recent Advances in theSynthesis and Applications of Radiolabeled Compounds for Drug Discoveryand Development. [In: Curr., Pharm. Des., 2000; 6(10)] 2000, 110 pp;George W.; Varma, Rajender S. The Synthesis of Radiolabeled Compoundsvia Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21;and Evans, E. Anthony. Synthesis of radiolabeled compounds, J.Radioanal. Chem., 1981, 64(1-2), 9-32.

Deuterated starting materials are readily available and are subjected tothe synthetic methods described herein to provide for the synthesis ofdeuterium-containing compounds. Large numbers of deuterium-containingreagents and building blocks are available commercially from chemicalvendors, such as Aldrich Chemical Co.

Deuterium-transfer reagents suitable for use in nucleophilicsubstitution reactions, such as iodomethane-d₃ (CD₃I), are readilyavailable and may be employed to transfer a deuterium-substituted carbonatom under nucleophilic substitution reaction conditions to the reactionsubstrate. The use of CD₁I is illustrated, by way of example only, inthe reaction schemes below.

Deuterium-transfer reagents, such as lithium aluminum deuteride(LiAlD₄), are employed to transfer deuterium under reducing conditionsto the reaction substrate. The use of LiAlD₄ is illustrated, by way ofexample only, in the reaction schemes below.

Deuterium gas and palladium catalyst are employed to reduce unsaturatedcarbon-carbon linkages and to perform a reductive substitution of arylcarbon-halogen bonds as illustrated, by way of example only, in thereaction schemes below.

In one embodiment, the compounds disclosed herein contain one deuteriumatom. In another embodiment, the compounds disclosed herein contain twodeuterium atoms. In another embodiment, the compounds disclosed hereincontain three deuterium atoms. In another embodiment, the compoundsdisclosed herein contain four deuterium atoms. In another embodiment,the compounds disclosed herein contain five deuterium atoms. In anotherembodiment, the compounds disclosed herein contain six deuterium atoms.In another embodiment, the compounds disclosed herein contain more thansix deuterium atoms. In another embodiment, the compound disclosedherein is fully substituted with deuterium atoms and contains nonon-exchangeable ¹H hydrogen atoms. In one embodiment, the level ofdeuterium incorporation is determined by synthetic methods in which adeuterated synthetic building block is used as a starting material.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts. A pharmaceutically acceptable salt of any one of the opioidreceptor antagonist prodrug compounds described herein is intended toencompass any and all pharmaceutically suitable salt forms. Preferredpharmaceutically acceptable salts of the compounds described herein arepharmaceutically acceptable acid addition salts and pharmaceuticallyacceptable base addition salts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid,hydrofluoric acid, phosphorous acid, and the like. Also included aresalts that are formed with organic acids such as aliphatic mono- anddicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoicacids, alkanedioic acids, aromatic acids, aliphatic and aromaticsulfonic acids, etc. and include, for example, acetic acid,trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like. Exemplary salts thus include sulfates,pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates,trifluoroacetates, propionates, caprylates, isobutyrates, oxalates,malonates, succinate suberates, sebacates, fumarates, maleates,mandelates, benzoates, chlorobenzoates, methylbenzoates,dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates,phenylacetates, citrates, lactates, malates, tartrates,methanesulfonates, and the like. Also contemplated are salts of aminoacids, such as arginates, gluconates, and galacturonates (see, forexample, Berge S. M. et al., “Pharmaceutical Salts,” Journal ofPharmaceutical Science, 66:1-19 (1997)). Acid addition salts of basiccompounds are, in some embodiments, prepared by contacting the five baseforms with a sufficient amount of the desired acid to produce the saltaccording to methods and techniques with which a skilled artisan isfamiliar.

“Pharmaceutically acceptable base addition salt” refers to those saltsthat retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Pharmaceutically acceptable base addition salts are, insome embodiments, formed with metals or amines, such as alkali andalkaline earth metals or organic amines. Salts derived from inorganicbases include, but are not limited to, sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amities and basic ion exchange resins, for example,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine,hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline,N-methylglucamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. See Berge et al., supra.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to therapeutic benefit and/or a prophylactic benefit. By“therapeutic benefit” is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient is still afflicted with the underlying disorder. Forprophylactic benefit, the compositions are, in some embodiments,administered to a patient at risk of developing a particular disease, orto a patient reporting one or more of the physiological symptoms of adisease, even though a diagnosis of this disease has not been made.

Opioid Receptor Pharmacology

The opioid receptors, μ, δ, κ, and the opioid-like receptor ORL-1 belongto the super family of G-protein coupled receptors (GPCRs) that possessseven helical trans-membrane spanning domains in their architecture. Themajority of research efforts focused upon this group of proteins hasbeen directed toward the μ receptor since it mediates the actions ofboth the opiate and opioid analgesics such as morphine and fentanyl,respectively. However, over the years it has become increasingly clearthat the entire family of proteins is actively involved in a host ofbiological processes. Furthermore, the advent of selective antagonistshas demonstrated that pharmacotherapeutic opportunities exist via bothnegative and positive modulation of this receptor family.

The μ (mu, OP₃ or MOP) receptor was originally defined and characterizedpharmacologically by Martin, Kosterlitz and their colleagues on thebasis of its high affinity for, and sensitivity to, morphine (Martin etal. The effects of morphine- and nalorphine-like drugs in thenondependent and morphine-dependent chronic spinal dog J. Pharmacol.Exp. Ther. (1976), 197: 517-532; Kosterlitz, et al. Endogenous opioidpeptides: multiple agonists and receptors, Nature (1977) 267: 495-499).The endogenous opioids, [Met⁵]-enkephalin, [Leu⁵]-enkephalin, extendedforms of [Met⁵]-enkephalin including metorphamide and BAM-18,β-endorphin, and truncated forms of dynorphin (e.g. dynorphin-(1-9) andshorter dynorphin peptides), also have affinities for μ receptors thatare consistent with a possible role for each of these peptides asnatural ligands for this receptor type, although these endogenouspeptides are not selective for μ receptors. Two putative naturalligands, endonamphin-1 and -2, that appear to mediate their effectsexclusively through the μ opioid receptor, also have been reported to bepresent in brain although no gene, precursor protein, or other mechanismfor their endogenous synthesis has been identified.

The μ receptors are distributed throughout the neuraxis. The highest μreceptor densities are found in the thalamus, caudate putamen,neocortex, nucleus accumbens, amygdala, interpeduncular complex, andinferior and superior colliculi (Watson et al. Autoradiographicdifferentiation of mu, delta and kappa receptors in the rat forebrainand midbrain, J. Neurosci. (1987), 7: 2445-2464). Then receptors, aswell as δ and κ receptors, are also present in the superficial layers ofthe dorsal horn of spinal cord. A moderate density of μ receptors isfound in periaqueductal gray and raphé nuclei. These brain regions havea well-established role in pain and analgesia. Other physiologicalfunctions regulated by μ receptors include respiratory andcardiovascular functions, intestinal transit, feeding, mood,thermoregulation, hormone secretion and immune functions.

The δ (delta, OP₁ or DOP) opioid receptor was defined using the mousevas deferens preparation and the enkephalins are generally consideredthe preferred endogenous ligands. The δ receptors are discretelydistributed in the central nervous system (CNS), with a prominentgradient of receptor density from high levels in forebrain structures torelatively low levels in most hindbrain regions. The highest densitiesare found in olfactory bulb, neocortex, caudate putamen, nucleusaccumbens, and amygdala (Watson et al. Autoradiographic differentiationof mu, delta and kappa receptors in the rat forebrain and midbrain, J.Neurosci. (1987), 7: 2445-2464). The thalamus and hypothalamus have amoderate density of δ receptors; in more caudal regions theinterpeduncular nucleus and pontine nuclei show high binding in rat, butmuch lower levels in mouse (Kitchen et al. Quantitative autoradiographicmapping of mu, delta and kappa-opioid receptors in knockout mice lackingthe mu-opioid receptor gene, Brain Res. (1997), 778: 73-88). In thespinal cord, δ receptors are present in dorsal horn where they play arole in mediating the analgesic effects of δ agonists.

The κ (kappa, OP₂ or KOP) opioid receptor was first proposed on thebasis of in vivo studies in dogs with ketocyclazocine and related drugs(Martin et al. The effects of morphine- and nalorphine-like drugs in thenondependent and morphine-dependent chronic spinal dog J. Pharmacol.Exp. Ther. (1976), 197: 517-532). Subsequent studies have confirmed thepresence of this receptor type in other species including guinea pig, aspecies that was preferred for many of the early studies on kappa opioidreceptors. Dynorphins A and B and α-neoendorphin appear to be theendogenous ligands for opioid κ receptors, although shorter peptidesderived from prodynorphin have comparable affinities at μ and κreceptors. The κ receptors are located predominantly in the cerebralcortex, nucleus accumbens, claustrum and hypothalamus of rat and mouse(Kitchen et al. Quantitative autoradiographic mapping of mu, delta andkappa-opioid receptors in knockout mice lacking the mu-opioid receptorgene, Brain Res. (1997), 778: 73-88; Watson et al. Autoradiographicdifferentiation of mu, delta and kappa receptors in the rat forebrainand midbrain, J. Neurosci. (1987), 7: 2445-2464), and have beenimplicated in the regulation of nociception, diuresis, feeding,neuroendocrine and immune system functions (Dhawan et al. InternationalUnion of Pharmacology, XII. Classification for opioid receptors,Pharmacol. Rev. (1996), 48: 567-592).

ORL1 receptors (also called nociceptin, or orphaninFQ receptors) are theyoungest members of the opioid receptor family. Agonist-inducedinternalization of ORL1 is rapid and concentration dependent. Agonistchallenge also reduces the ability of ORL1 to couple to inhibition offorskolin-stimulated cAMP production, suggesting that ORL1 undergoessimilar desensitization mechanisms as compared with the other threeopioid receptors subtypes.

The structure of the ORL1 receptor indicates that it has evolved as partof the opioid receptor family. Sequence comparisons with μ, κ, and δreceptors, and with other similar G protein-coupled receptors (e.g. ofthe SOM receptor family), indicate that the ORL1 receptor is moreclosely related to opioid receptors than to other types of Gprotein-coupled receptors (Birgul, et al. Reverse Physiology indrosophila: identification of a novel allatostatin-like neuropeptide andits cognate receptor structurally related to the mammaliansomatostatin/galanin/opioid receptor family, EMBO J. (1999), 18:5892-5900). Additionally, agonists at ORL1 receptors induce activationof the same set of transduction pathways activated by μ, κ, and δreceptors, and the endogenous ligand, ORL1, shares considerable sequencehomology with dynorphin A and, to a lesser extent, with the enkephalins.Thus, the ORL1 receptor and its endogenous ligand are closely related inan evolutionary sense to the μ, κ, and δ receptors.

Despite the evidence of evolutionary and functional homology, the ORL1receptor is not an opioid receptor from a pharmacological perspective.The effects of activation of this receptor are not obviously‘opiate-like’ with respect to pain perception. The ORL1 receptor hasnegligible affinity for naloxone and for most other antagonists at μ, κor δ receptors. The ORL1 receptor is, however, expressed in manyfunctional systems in which endogenous opioids play a regulatory role.Although the functions of ORL1 are not yet filly understood, regulatoryfunctions for ORL1 parallel to but not identical to those of theendogenous opioid peptides seem very probable. Despite these functionaldifferences, the subcommittee finds the structural relationship betweenthe ORL1 receptor and μ, δ and κ receptors compelling.

ORL1 receptor regulation, while increasingly studied, is still in theinfant stages of understanding when compared to the other three opioidreceptor subtypes. To date few site-directed mutagenesis studies havebeen conducted, and receptor regulation in primary neurons, dorsal rootganglion, or dorsal horn neurons remains unknown.

An integral part of the effort to characterize the opioid receptorsystem has been the discovery of potent, pure antagonists of opioidreceptors. Nalmefene (1a) and naltrexone (1b), both competitiveantagonists at μ, δ, and κ opioid receptors, were used aspharmacological tools to identify and characterize opioid systems.

Nalmefene is an opioid receptor antagonist that has been available forseveral years as Revex® injection for use in reversing opioid effectsand for opioid overdose. Nalmefene is also described in literature forthe treatment of substance abuse disorders such as alcohol dependenceand abuse, and impulse control disorders such as pathological gamblingand addiction to shopping. It is marketed as Selincro in Europe as an ondemand oral pill for alcohol abuse. It has the IUPAC name17-cyclopropylmethyl-4,5α-epoxy-6-methylenemorphinan-3,14-diol and hasthe structure provided in Formula (1A).

Naltrexone is an opioid receptor antagonist used primarily in themanagement of alcohol dependence and opioid dependence. It is marketedin the generic form as its hydrochloride salt, naltrexone hydrochlorideunder the trade names Revia® and Depade® in the form of 50 mg filmcoated tablets. Once monthly extended release naltrexone, marketed inthe United States as Vivitrol, has gained wide acceptance in opioid usedisorder due to increased patient adherence. Naltrexone has the IUPACname 17-(cyclopropylmethyl)-4,5α-epoxy-3,14-dihydroxymorphinan-6-one andhas the structure provided in Formula (1B)

Low doses of naltrexone have also been investigated in patients withmultiple sclerosis, autism, active Crohn's disease, AIDS, rheumatoidarthritis, celiac disease, certain forms of cancer, and autoimmunediseases. Opioids act as cytokines, the principal communicationsignallers of the immune system, creating immunomodulatory effectsthrough opioid receptors on immune cells. Very low doses of naltrexonewere shown to boost the immune system and helps to fight againstdiseases characterized by inadequate immune function.

In terms of pharmacology, naltrexone blocks the effects of opioids byits highly competitive binding at the μ-opioid receptors. Being acompetitive antagonist, the suppression of an opiate's agonistic,euphorigenic effect can be overcome. However, clinical studies haveindicated that naltrexone in an oral dosage of approximately 50 mg isable to block the pharmacological effects of up to 25 mg ofintravenously administered heroin for periods as long as twenty fourhours.

The mechanism of action of naltrexone in the treatment of alcoholism isnot understood although involvement of the endogenous opioid system issuggested by preclinical data. Opioid antagonists have been shown toreduce alcohol consumption by animals, and naltrexone has shown efficacyin maintaining abstinence in clinical studies in humans.

Opioid Receptor Antagonists Prodrugs

Although using nalmefene and naltrexone in the treatment of alcoholdependence and opioid dependence provides a great benefit to thesociety, the problem with these drugs is that they have very shortperiod of action. Thus, for example, well absorbed orally (approximately96% of an oral dose is absorbed from the gastrointestinal tract),naltrexone is subject to significant first pass metabolism with oralbioavailability estimates ranging from 5% to 40%. The activity ofnaltrexone is believed to be as a result of both naltrexone and its6-β-naltrexol metabolite. Two other minor metabolites are2-hydroxy-3-methoxy-6-β-naltrexol and 2-hydroxy-3-methyl-naltrexone.Peak plasma levels of both naltrexone and 6-β-naltexol occur within onehour after oral dosing; mean elimination half-life values for naltrexoneand 6-β-naltrexol are four and thirteen hours respectively. Even forlong acting naltrexone injections, clinicians indicate that patientsdiscontinue treatment too early. Therefore, a need exists for ultra-longacting opioid antagonists in the treatment of substance abuse disorder.

One of the solutions to overcome the problem of short period of actionof nalmefene and naltrexone is to use prodrugs which provide a long,sustained, and controlled release of nalmefene and naltrexone opioidreceptor antagonists upon administration into the body.

As used in this disclosure, the term “prodrug” is meant to indicate acompound that is converted under physiological conditions to nalmefeneor naltrexone. A prodrug, in some embodiments, is inactive whenadministered to a subject, but is converted in vivo to an activecompound, for example, by hydrolysis. Thus, the term “prodrug” refers toa precursor compound that is pharmaceutically acceptable, and in someembodiments, is devoid of the pharmacological properties of nalmefene ornaltrexone. The prodrug compound often offers advantages of solubility,tissue compatibility or delayed release in a mammalian organism (see,e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier,Amsterdam).

A discussion of prodrugs is provided in Higuchi, T., et al., “Pro-drugsas Novel Delivery Systems,” A.C.S. Symposium Series, Vol. 14, and inBioreversible Carriers in Drug Design, ed, Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound in vivo when such prodrug isadministered to a mammalian subject. Prodrugs of nalmefene ornaltrexone, as described herein, are prepared by modifying functionalgroups present in the active compound in such a way that themodifications are cleaved to the parent active compound. Prodrugsinclude compounds wherein a hydroxy group is bonded to any group that,when the prodrug of the active compound is administered to a mammaliansubject, cleaves to form a free hydroxy group.

Provided herein are prodrugs of opioid receptor antagonists nalmefeneand naltrexone.

In one aspect, provided herein is a compound, or pharmaceuticallyacceptable salt thereof, having a structure provided in Formula (I),

wherein,

X is O or CH₂;

R is selected from:

a. (C₃-C₇cycloalkyl)CH₂C(O)—;

b. (C₃-C₇cycloalkyl)CH₂CH₂C(O)—;

c. —C(O)OC₇-C₂₀ alkyl; or

d. —C(O)NHC(CH₃)₃.

In some embodiments, X is O. In some embodiments, X is CH₂.

In some embodiments, R is (C₃-C₇cycloalkyl)CH₂C(O)—. In someembodiments, R is (C₃-C₇cycloalkyl)CH₂CH₂C(O)—. In some embodiments, Ris —C(O)OC₇-C₂₀ alkyl. In some embodiments, R is —C(O)NHC(CH₃)₃.

In some embodiments, R is (C₃-C₇cycloalkyl)CH₂C(O)—. In someembodiments, R is (C₃-C₄cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₃-C₅cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₃-C₆cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₄-C₅cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₄-C₆cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₄-C₇cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₅-C₆cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₅-C₇cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₆-C₇cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₃cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₄cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₅cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₆cycloalkyl)CH₂C(O)—. In some embodiments, R is(C₇cycloalkyl)CH₂C(O)—.

In some embodiments, R is (C₃-C₇cycloalkyl)CH₂CH₂C(O)—. In someembodiments, R is (C₃-C₄cycloalkyl)CH₂CH₂C(O)—. In some embodiments, Ris (C₃-C₅cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₃-C₆cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₄-C₅cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₄-C₆cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₄-C₇cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₅-C₆cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₅-C₇cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₆-C₇cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₃cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₄cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₅cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₆cycloalkyl)CH₂CH₂C(O)—. In some embodiments, R is(C₇cycloalkyl)CH₂CH₂C(O)—.

In some embodiments, R is —C(O)OC₇-C₂₀ alkyl. In some embodiments, R is—C(O)OC₇-C₈ alkyl. In some embodiments, R is —C(O)OC₇-C₉ alkyl. In someembodiments, R is —C(O)OC₇-C₁₀ alkyl. In some embodiments, R is—C(O)OC₇-C₁₁ alkyl. In some embodiments, R is —C(O)OC₇-C₁₂ alkyl. Insome embodiments, R is —C(O)—OC₇-C₁₃ alkyl. In some embodiments, R is—C(O)OC₇-C₁₄ alkyl. In some embodiments, R is —C(O)OC₇-C₁₅ alkyl. Insome embodiments, R is —C(O)OC₇-C₁₆ alkyl. In some embodiments, R is—C(O)OC₇-C₁₇ alkyl. In some embodiments, R is —C(O)OC₇-C₁₈ alkyl. Insome embodiments, R is —C(O)OC₇-C₁₉ alkyl. In some embodiments, R is—C(O)OC₈-C₉ alkyl. In some embodiments, R is —C(O)OC₈-C₁₀ alkyl. In someembodiments, R is —C(O)OC₈-C₁₁ alkyl. In some embodiments, R is—C(O)OC₈-C₁₂ alkyl. In some embodiments, R is —C(O)OC₈-C₁₃ alkyl. Insome embodiments, R is —C(O)OC₈-C₁₄ alkyl. In some embodiments, R is—C(O)OC₈-C₁₅ alkyl. In some embodiments, R is —C(O)OC₈-C₁₆ alkyl. Insome embodiments, R is —C(O)OC₈-C₁₇ alkyl. In some embodiments, R is—C(O)OC₈-C₁₈ alkyl. In some embodiments, R is —C(O)OC₈-C₁₉ alkyl. Insome embodiments, R is —C(O)OC₈-C₂₀ alkyl. In some embodiments, R is—C(O)OC₉-C₁₀ alkyl. In some embodiments, R is —C(O)OC₉-C₁₁ alkyl. Insome embodiments, R is —C(O)OC₉-C₁₂ alkyl. In some embodiments, R is—C(O)OC₉-C₁₃ alkyl. In some embodiments, R is —C(O)OC₉-C₁₄ alkyl. Insome embodiments, R is —C(O)OC₉-C₁₅ alkyl. In some embodiments, R is—C(O)OC₉-C₁₆ alkyl. In some embodiments, R is —C(O)OC₉-C₁₇ alkyl. Insome embodiments, R is —C(O)OC₉-C₁₈ alkyl. In some embodiments, R is—C(O)OC₉-C₁₉ alkyl. In some embodiments, R is —C(O)OC₉-C₂₀ alkyl. Insome embodiments, R is —C(O)OC₁₀-C₁₁ alkyl. In some embodiments, R is—C(O)OC₁₀-C₁₂ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₃ alkyl. Insome embodiments, R is —C(O)OC₁₀-C₁₄ alkyl. In some embodiments, R is—C(O)OC₁₀-C₁₅ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₆ alkyl. Insome embodiments, R is —C(O)OC₁₀-C₁₇ alkyl. In some embodiments, R is—C(O)OC₁₀-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₀-C₁₉ alkyl. Insome embodiments, R is —C(O)OC₁₀-C₂₀ alkyl. In some embodiments, R is—C(O)OC₁₁-C₁₂ alkyl. In some embodiments, R is —C(O)OC₁₁-C₁₃ alkyl. Insome embodiments, R is —C(O)OC₁₁-C₁₄ alkyl. In some embodiments, R is—C(O)OC₁₁-C₁₅ alkyl. In some embodiments, R is —C(O)OC₁₁-C₁₆ alkyl. Insome embodiments, R is —C(O)OC₁₁-C₁₇ alkyl. In some embodiments, R is—C(O)OC₁₁-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₁-C₁₉ alkyl. Insome embodiments, R is —C(O)OC₁₁-C₂₀ alkyl. In some embodiments, R is—C(O)OC₁₂-C₁₃ alkyl. In some embodiments, R is —C(O)OC₁₂-C₁₄ alkyl. Insome embodiments, R is —C(O)OC₁₂-C₁₅ alkyl. In some embodiments, R is—C(O)OC₁₂-C₁₆ alkyl. In some embodiments, R is —C(O)OC₁₂-C₁₇ alkyl. Insome embodiments, R is —C(O)OC₁₂-C₁₈ alkyl. In some embodiments, R is—C(O)OC₁₂-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₂-C₂₀ alkyl. Insome embodiments, R is —C(O)OC₁₃-C₁₄ alkyl. In some embodiments, R is—C(O)OC₁₃-C₁₅ alkyl. In some embodiments, R is —C(O)OC₁₃-C₁₆ alkyl. Insome embodiments, R is —C(O)OC₁₃-C₁₇ alkyl. In some embodiments, R is—C(O)OC₁₃-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₃-C₁₉ alkyl. Insome embodiments, R is —C(O)OC₁₃-C₂₀ alkyl. In some embodiments, R is—C(O)OC₁₄-C₁₅ alkyl. In some embodiments, R is —C(O)OC₁₄-C₁₆ alkyl. Insome embodiments, R is —C(O)OC₁₄-C₁₇ alkyl. In some embodiments, R is—C(O)OC₁₄-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₄-C₁₉ alkyl. Insome embodiments, R is —C(O)OC₁₄-C₂₀ alkyl. In some embodiments, R is—C(O)OC₁₅-C₁₆ alkyl. In some embodiments, R is —C(O)OC₁₅-C₁₇ alkyl. Insome embodiments, R is —C(O)OC₁₅-C₁₈ alkyl. In some embodiments, R is—C(O)OC₁₅-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₅-C₂₀ alkyl. Insome embodiments, R is —C(O)OC₁₆-C₁₇ alkyl. In some embodiments, R is—C(O)OC₁₆-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₆-C₁₉ alkyl. Insome embodiments, R is —C(O)OC₁₆-C₂₀ alkyl. In some embodiments, R is—C(O)OC₁₇-C₁₈ alkyl. In some embodiments, R is —C(O)OC₁₇-C₁₉ alkyl. Insome embodiments, R is —C(O)OC₁₇-C₂₀ alkyl. In some embodiments, R is—C(O)OC₁₈-C₁₉ alkyl. In some embodiments, R is —C(O)OC₁₈-C₂₀ alkyl. Insome embodiments, R is —C(O)OC₁₉-C₂₀ alkyl. In some embodiments, R is—C(O)OC₇ alkyl. In some embodiments, R is —C(O)OC₈ alkyl. In someembodiments, R is —C(O)OC₉ alkyl. In some embodiments, R is —C(O)OC₁₀alkyl. In some embodiments, R is —C(O)OC₁₁ alkyl. In some embodiments, Ris —C(O)OC₁₂ alkyl. In some embodiments, R is —C(O)OC₁₃ alkyl. In someembodiments, R is —C(O)OC₁₄ alkyl. In some embodiments, R is —C(O)OC₁₅alkyl. In some embodiments, R is —C(O)OC₁₆ alkyl. In some embodiments, Ris —C(O)OC₁₇ alkyl. In some embodiments, R is —C(O)OC₁₈ alkyl. In someembodiments, R is —C(O)OC₁₉ alkyl. In some embodiments, R is —C(O)OC₂₀alkyl.

In some embodiments, R is —C(O)NHC(CH₃)₃.

In another aspect, also provided herein is a compound, orpharmaceutically acceptable salt thereof, having a structure provided inFormula (II),

wherein,

X is O or CH₂;

R is:

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl; and n is 7-15;provided if X is O, then n is not 7.

In some embodiments, X is O. In some embodiments, X is CH₂.

In some embodiments, R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl.

In some embodiments, R¹ is a C₄-C₁₀ alkyl. In some embodiments, R¹ is aC₄-C₅ alkyl. In some embodiments, R¹ is a C₄-C₆ alkyl. In someembodiments, R¹ is a C₄-C₇ alkyl. In some embodiments, R¹ is a C₄-C₈alkyl. In some embodiments, R¹ is a C₄-C₉ alkyl. In some embodiments, R¹is a C₅-C₆ alkyl. In some embodiments, R¹ is a C₅-C₇ alkyl. In someembodiments, R¹ is a C₅-C₈ alkyl. In some embodiments, R¹ is a C₅-C₉alkyl. In some embodiments, R¹ is a C₅-C₁₀ alkyl. In some embodiments,R¹ is a C₆-C₇ alkyl. In some embodiments, R¹ is a C₆-C₈ alkyl. In someembodiments, R¹ is a C₆-C₉ alkyl. In some embodiments, R¹ is a C₆-C₁₀alkyl In some embodiments, R¹ is a C₇-C₈ alkyl. In some embodiments, R¹is a C₇-C₉ alkyl. In some embodiments, R¹ is a C₇-C₁₀ alkyl. In someembodiments, R¹ is a C₈-C₉ alkyl. In some embodiments, R¹ is a C₈-C₁₀alkyl. In some embodiments, R¹ is a C₉-C₁₀ alkyl. In some embodiments,R¹ is a C₄ alkyl. In some embodiments, R¹ is a C₅ alkyl. In someembodiments, R¹ is a C₆ alkyl. In some embodiments, R¹ is a C₇ alkyl. Insome embodiments, R¹ is a C₈ alkyl. In some embodiments, R¹ is a C₉alkyl. In some embodiments, R¹ is a C₁₀ alkyl.

In some embodiments, R¹ is a C₄-C₁₀ alkenyl. In some embodiments, R¹ isa C₄-C₅ alkenyl. In some embodiments, R¹ is a C₄-C₆ alkenyl. In someembodiments, R¹ is a C₄-C₇ alkenyl. In some embodiments, R¹ is a C₄-C₈alkenyl. In some embodiments, R¹ is a C₄-C₉ alkenyl. In someembodiments, R¹ is a C₅-C₆ alkenyl. In some embodiments, R¹ is a C₅-C₇alkenyl. In some embodiments, R¹ is a C₅-C₈ alkenyl. In someembodiments, R¹ is a C₅-C₉ alkenyl. In some embodiments, R¹ is a C₅-C₁₀alkenyl. In some embodiments, R¹ is a C₆-C₇ alkenyl. In someembodiments, R¹ is a C₆-C₈ alkenyl. In some embodiments, R¹ is a C₆-C₉alkenyl. In some embodiments, R¹ is a C₆-C₁₀ alkenyl. In someembodiments, R¹ is a C₇-C₈ alkenyl. In some embodiments, R¹ is a C₇-C₉alkenyl. In some embodiments, R¹ is a C₇-C₁₀ alkenyl. In someembodiments, R¹ is a C₈-C₉ alkenyl. In some embodiments, R¹ is a C₈-C₁₀alkenyl. In some embodiments, R¹ is a C₉-C₁₀ alkenyl. In someembodiments, R¹ is a C₄ alkenyl. In some embodiments, R¹ is a C₅alkenyl. In some embodiments, R¹ a C₆ alkenyl. In some embodiments, R¹is a C₇ alkenyl. In some embodiments, R¹ is a C₈ alkenyl. In someembodiments, R¹ is a C₉ alkenyl. In some embodiments, R¹ is a C₁₀alkenyl.

In some embodiments, n is 7-15. In some embodiments, n is 7-8. In someembodiments, n is 7-9. In some embodiments, n is 7-10. In someembodiments, n is 7-11. In some embodiments, n is 7-12. In someembodiments, n is 7-13. In some embodiments, n is 7-14. In someembodiments, n is 8-9. In some embodiments, n is 8-10. In someembodiments, n is 8-11. In some embodiments, n is 8-12. In someembodiments, n is 8-13. In some embodiments, n is 8-14. In someembodiments, n is 8-15. In some embodiments, n is 9-10. In someembodiments, n is 9-11. In some embodiments, n is 9-12. In someembodiments, n is 9-13. In some embodiments, n is 9-14. In someembodiments, n is 9-15. In some embodiments, n is 10-11. In someembodiments, n is 10-12. In some embodiments, n is 10-13. In someembodiments, n is 10-14. In some embodiments, n is 10-15. In someembodiments, n is 11-12. In some embodiments, n is 11-13. In someembodiments, n is 11-14. In some embodiments, n is 11-15. In someembodiments, n is 12-13. In some embodiments, n is 12-14. In someembodiments, n is 12-15. In some embodiments, n is 13-14. In someembodiments, n is 13-15, some embodiments, n is 14-15. In someembodiments, n is 7. In some embodiments, n is 8. In some embodiments, nis 9. In some embodiments, n is 10. In some embodiments, n is 11. Insome embodiments, n is 12. In some embodiments, n is 13. In someembodiments, n is 14. In some embodiments, n is 15.

In another aspect, also provided herein is a compound, orpharmaceutically acceptable salt thereof, having a structure provided inFormula (II),

wherein,

X is O or CH₂;

R is

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl; and n is 7-15;provided if X is O, then n is not 7.

In some embodiments, X is O. In some embodiments, X is CH₂.

In some embodiments, R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl.

In some embodiments, R¹ is a C₄-C₁₀ alkyl. In some embodiments, R¹ is aC₄-C₅ alkyl. In some embodiments, R¹ is a C₄-C₆ alkyl. In someembodiments, R¹ is a C₄-C₇ alkyl. In some embodiments, R¹ is a C₄-C₈alkyl. In some embodiments, R¹ is a C₄-C₉ alkyl. In some embodiments, R¹is a C₅-C₆ alkyl. In some embodiments, R¹ is a C₅-C₇ alkyl. In someembodiments, R¹ is a C₅-C₈ alkyl. In some embodiments, R¹ is a C₅-C₉alkyl. In some embodiments, R¹ is a C₅-C₁₀ alkyl. In some embodiments,R¹ is a C₆-C₇ alkyl. In some embodiments, R¹ is a C₆-C₈ alkyl. In someembodiments, R¹ is a C₆-C₉ alkyl. In some embodiments, R¹ is a C₆-C₁₀alkyl. In some embodiments, R¹ is a C₇-C₈ alkyl. In some embodiments, R¹is a C₇-C₉ alkyl. In some embodiments, R¹ is a C₇-C₁₀ alkyl. In someembodiments, R¹ is a C₈-C₉ alkyl. In some embodiments, R¹ is a C₈-C₁₀alkyl. In some embodiments, R¹ is a C₉-C₁₀ alkyl. In some embodiments,R¹ is a C₄ alkyl. In some embodiments, R¹ is a C₅ alkyl. In someembodiments, R¹ is a C₆ alkyl. In some embodiments, R¹ is a C₇ alkyl. Insome embodiments, R¹ is a C₈ alkyl. In some embodiments, R¹ is a C₉alkyl. In some embodiments, R¹ is a C₁₀ alkyl.

In some embodiments, R¹ is a C₄-C₁₀ alkenyl. In some embodiments, R¹ isa C₄-C₅ alkenyl. In some embodiments, R¹ is a C₄-C₆ alkenyl. In someembodiments, R¹ is a C₄-C₇ alkenyl. In some embodiments, R¹ is a C₄-C₈alkenyl. In some embodiments, R¹ is a C₄-C₉ alkenyl. In someembodiments, R¹ is a C₅-C₆ alkenyl. In some embodiments, R¹ is a C₅-C₇alkenyl. In some embodiments, R¹ is a C₅-C₈ alkenyl. In someembodiments, R¹ is a C₅-C₉ alkenyl. In some embodiments, R¹ is a C₅-C₁₀alkenyl. In some embodiments, R¹ is a C₆-C₇ alkenyl. In someembodiments, R¹ is a C₆-C₈ alkenyl. In some embodiments, R¹ is a C₆-C₉alkenyl. In some embodiments, R¹ is a C₆-C₁₀ alkenyl. In someembodiments, R¹ is a C₇-C₈ alkenyl. In some embodiments, R¹ is a C₇-C₉alkenyl. In some embodiments, R¹ is a C₇-C₁₀ alkenyl. In someembodiments, R¹ is a C₈-C, alkenyl. In some embodiments, R¹ is a C₈-C₁₀alkenyl. In some embodiments, R¹ is a C₉-C₁₀ alkenyl. In someembodiments, R¹ is a C₄ alkenyl. In some embodiments, R¹ is a C₅alkenyl. In some embodiments, R¹ is a C₆ alkenyl. In some embodiments,R¹ is a C₇ alkenyl. In some embodiments, R¹ is a C₈ alkenyl. In someembodiments, R¹ is a C₉ alkenyl. In some embodiments, R¹ is a C₁₀alkenyl.

In some embodiments, n is 7-15. In some embodiments, n is 7-8. In someembodiments, n is 7-9. In some embodiments, n is 7-10. In someembodiments, n is 7-11. In some embodiments, n is 7-12. In someembodiments, n is 7-13. In some embodiments, n is 7-14. In someembodiments, n is 8-9. In some embodiments, n is 8-10. In someembodiments, n is 8-11. In some embodiments, n is 8-12. In someembodiments, n is 8-13. In some embodiments, n is 8-14. In someembodiments, n is 8-15. In some embodiments, n is 9-10. In someembodiments, n is 9-11. In some embodiments, n is 9-12. In someembodiments, n is 9-13. In some embodiments, n is 9-14. In someembodiments, n is 9-15. In some embodiments, n is 10-11. In someembodiments, n is 10-12. In some embodiments, n is 10-13. In someembodiments, n is 10-14. In some embodiments, n is 10-15. In someembodiments, n is 11-12. In some embodiments, n is 11-13. In someembodiments, n is 11-14. In some embodiments, n is 11-15. In someembodiments, n is 12-13. In some embodiments, n is 12-14. In someembodiments, n is 12-15. In some embodiments, n is 13-14. In someembodiments, n is 13-15. In some embodiments, n is 14-15. In someembodiments, n is 7. In some embodiments, n is 8. In some embodiments, nis 9. In some embodiments, n is 10. In some embodiments, n is 11. Insome embodiments, n is 12. In some embodiments, n is 13. In someembodiments, n is 14. In some embodiments, n is 15.

In another aspect, also provided herein is a compound, orpharmaceutically acceptable salt thereof, having a structure provided inFormula (IIa),

wherein,

X is O or CH₂;

R is:

wherein R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl; and n is 9-15.

In some embodiments, X is O. In some embodiments, X is CH₂.

In some embodiments, R¹ is a C₄-C₁₀ alkyl or a C₄-C₁₀ alkenyl.

In some embodiments, R¹ is a C₄-C₁₀ alkyl. In some embodiments, R¹ is aC₄-C₅ alkyl. In some embodiments, R¹ is a C₄-C₆ alkyl. In someembodiments, R¹ is a C₄-C₇ alkyl. In some embodiments, R¹ is a C₄-C₈alkyl. In some embodiments, R¹ is a C₄-C₉ alkyl. In some embodiments, R¹is a C₅-C₆ alkyl. In some embodiments, R¹ is a C₅-C₇ alkyl. In someembodiments, R¹ is a C₅-C₈ alkyl. In some embodiments, R¹ is a C₅-C₉alkyl. In some embodiments, R¹ is a C₅-C₁₀ alkyl. In some embodiments,R¹ is a C₆-C₇ alkyl. In some embodiments, R¹ is a C₆-C₈ alkyl. In someembodiments, R¹ is a C₆-C₉ alkyl. In some embodiments, R¹ is a C₆-C₁₀alkyl. In some embodiments, R¹ is a C₇-C₈ alkyl. In some embodiments, R¹is a C₇-C₉ alkyl. In some embodiments, R¹ is a C₇-C₁₀ alkyl. In someembodiments, R¹ is a C₈-C₉ alkyl. In some embodiments, R¹ is a C₈-C₁₀alkyl. In some embodiments, R¹ is a C₉-C₁₀ alkyl. In some embodiments,R¹ is a C₄ alkyl. In some embodiments, R¹ is a C₅ alkyl. In someembodiments, R¹ is a C₆ alkyl. In some embodiments, R¹ is a C₇ alkyl. Insome embodiments, R¹ is a C₈ alkyl. In some embodiments, R¹ is a C₉alkyl. In some embodiments, R¹ is a C₁₀ alkyl.

In some embodiments, R¹ is a C₄-C₁₀ alkenyl. In some embodiments, R¹ isa C₄-C₅ alkenyl. In some embodiments, R¹ is a C₄-C₆ alkenyl. In someembodiments, R¹ is a C₄-C₇ alkenyl. In some embodiments, R¹ is a C₄-C₈alkenyl. In some embodiments, R¹ is a C₄-C₉ alkenyl. In someembodiments, R¹ is a C₅-C₆ alkenyl. In some embodiments, R¹ is a C₅-C₇alkenyl. In some embodiments, R¹ is a C₅-C₈ alkenyl. In someembodiments, R¹ is a C₅-C₉ alkenyl. In some embodiments, R¹ is a C₅-C₁₀alkenyl. In some embodiments, R¹ is a C₆-C₇ alkenyl. In someembodiments, R¹ is a C₆-C₈ alkenyl. In some embodiments, R¹ is a C₆-C₉alkenyl. In some embodiments, R¹ is a C₆-C₁₀ alkenyl. In someembodiments, R¹ is a C₇-C₈ alkenyl. In some embodiments, R¹ is a C₇-C₉alkenyl. In some embodiments, R¹ is a C₇-C₁₀ alkenyl. In someembodiments, R¹ is a C₈-C₉ alkenyl. In some embodiments, R¹ is a C₈-C₁₀alkenyl. In some embodiments, R¹ is a C₉-C₁₀ alkenyl. In someembodiments, R¹ is a C₄ alkenyl. In some embodiments, R¹ is a C₅alkenyl. In some embodiments, R¹ is a C₆ alkenyl. In some embodiments,R¹ is a C₇ alkenyl. In some embodiments, R¹ is a C₈ alkenyl. In someembodiments, R¹ is a C₉ alkenyl. In some embodiments, R¹ is a C₁₀alkenyl.

In some embodiments, n is 9-15. In some embodiments, n is 9-10. In someembodiments, n is 9-11. In some embodiments, n is 9-12. In someembodiments, n is 9-13. In some embodiments, n is 9-14. In someembodiments, n is 10-11. In some embodiments, n is 10-12. In someembodiments, n is 10-13. In some embodiments, n is 10-14. In someembodiments, n is 10-15. In some embodiments, n is 11-12. In someembodiments, n is 11-13. In some embodiments, n is 11-14. In someembodiments, n is 11-15. In some embodiments, n is 12-13. In someembodiments, n is 12-14. In some embodiments, n is 12-15. In someembodiments, n is 13-14. In some embodiments, n is 13-15. In someembodiments, n is 14-15. In some embodiments, n is 9. In someembodiments, n is 10. In some embodiments, n is 11. In some embodiments,n is 12. In some embodiments, n is 13. In some embodiments, n is 14. Insome embodiments, n is 15.

In another aspect, also provided herein is a compound, orpharmaceutically acceptable salt thereof, having a structure provided inFormula (III),

wherein,

X is O or CH₂;

R is selected from:

-   -   —[CH(R³)O]z-R⁴;    -   —[CH(R³)O]z-C(O)OR⁴;    -   —[CH(R³)O]z-C(═O)NR⁴R⁵; and    -   —[CH(R³)O]z-P(═O)(OR⁴)(OR⁵);    -   wherein z is 1, 2, 3, 4, 5, 6, or 7;    -   R³ is hydrogen, halogen, alkyl, alkenyl, cycloalkylalkyl, or        aryl;    -   each R⁴ and R⁵ is independently selected from hydrogen, alkyl,        alkenyl, cycloalkylalkyl, or aryl.

In some embodiments, X is O. In some embodiments, X is CH₂.

In some embodiments, z is 1. In some embodiments, z is 2. In someembodiments, z is 3. In some embodiments, z is 4. In some embodiments, zis 5. In some embodiments, z is 6. In some embodiments, z is 7. In someembodiments, z is 1 or 2. In some embodiments, z is 2 or 3. In someembodiments, z is 1, 2, or 3.

In some embodiments, R³ is hydrogen, halogen or alkyl. In someembodiments, R³ is alkyl. In some embodiments, R³ is hydrogen. In someembodiments, R³ is hydrogen, halogen, alkyl, cycloalkylalkyl, or aryl.In some embodiments, R³ is hydrogen, halogen, cycloalkylalkyl, or aryl.In some embodiments, R³ is halogen. In some embodiments, the halogen isfluorine.

In some embodiments, each R⁴ and R⁵ is independently selected fromalkyl, or aryl. In some embodiments, each R⁴ and R⁵ is independentlyselected from alkyl. In some embodiments, each R⁴ and R⁵ isindependently selected from hydrogen or alkyl. In some embodiments, thealkyl is C₁₀-C₁₈ alkyl. In some embodiments, the alkyl is C₅-C₉ alkyl.In some embodiments, the alkyl is C₁-C₄ alkyl. In some embodiments, thealkyl is C₉-C₁₃ alkyl. In some embodiments, the alkyl is C₁₀-C₁₂ alkyl.In some embodiments, the alkyl is C₁₀ alkyl. In some embodiments, thealkyl is C₁₁ alkyl. In some embodiments, the alkyl is C₁₂ alkyl.

In some embodiments, R is: —[CH(R³)O]z-R⁴. In some embodiments, R is:—[CH(R³)O]z-C(═O)OR⁴. In some embodiments, R is: —[CH(R³)O]z-C(═O)NR⁴R⁵.In some embodiments, R is: —[CH(R³)O]z-P(═O)(OR⁴)(OR⁵). In someembodiments, R is: —[CH(R³)O]z-C(═O)OR⁴, wherein R³ is hydrogen, and R⁴is C₉-C-₁₃ alkyl. In some embodiments, R is: —[CH(R³)O]z-C(═O)OR⁴,wherein R³ is hydrogen, and R⁴ is C₁₀-C₁₂ alkyl. In some embodiments, Ris: —[CH(R³)O]z-C(═O)OR⁴, wherein R³ is hydrogen, and R⁴ is C₁₀ alkyl.In some embodiments, R is: —[CH(R³)O]z-C(═O)OR⁴, wherein R³ is hydrogen,and R⁴ is C₁₁ alkyl. In some embodiments, R is: —[CH(R³)O]z-C(═O)OR⁴,wherein R³ is hydrogen, and R⁴ is C₁₂ alkyl,

In some embodiments, the opioid receptor antagonist prodrug compounddescribed herein has a structure provided in Table 1.

TABLE 1

Chemical Synthesis Example R X Chemical Name 1

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl((E)- octadec-9-en-1-yl) carbonate 2

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl undecyl carbonate 3

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl undecyl carbonate 4

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl undecyl carbonate 5

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl undecyl carbonate 6

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl dodecyl carbonate 7

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl((E)- octadec-9-en-1-yl) carbonate 8

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl(E)- octadec-9-enoate 9

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl(E)- octadec-9-enoate 10

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl decyl carbonate 11

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl dodecyl carbonate 12

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl stearate 13

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl(Z)- docos-13-enoate 14

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl docosanoate 15

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl(E)- octadec-9-enoate 16

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl icosanoate 17

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl octyl carbonate 18

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl decyl carbonate 19

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl hexadecyl carbonate 20

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl (9Z,12Z,15Z)-octadeca- 9,12,15-trienoate 21

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl hexadecyl carbonate 22

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl(Z)- docos-13-enoate 23

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl octyl carbonate 24

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl dodecyl carbonate 25

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl tetradecyl carbonate 26

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl(E)- octadec-9-enoate 27

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl tetradecyl carbonate 28

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl icosyl carbonate 29

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl dodecyl carbonate 30

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl tridecyl carbonate 31

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl tetradecyl carbonate 32

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl pentadecyl carbonate 33

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl octadecyl carbonate 34

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl hexadecyl carbonate 35

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl decyl carbonate 36

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl oleate 37

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl (9Z,12Z)-octadeca-9,12- dienoate 38

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl 3,3- dimethylbutanoate 39

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl 3- cyclopentylpropanoate 40

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl tert- butylcarbamate 41

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl oleate 42

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl 3,3- dimethylbutanoate 43

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl 3- cyclopentylpropanoate 44

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl dodecanoate 45

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl tetradecanoate 46

CH₂ (((4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl hexadecanoate 47

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl hexadecyl carbonate 48

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl dodecanoate 49

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl hexadecanoate

In some embodiments, the opioid receptor antagonist prodrug compounddescribed herein has a structure provided in Table 2.

TABLE 2

Example R X Chemical Name 50

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl icosanoate 51

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl docosanoate 52

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl (9Z,12Z)-octadeca- 9,12,15-trienoate 53

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl (9Z,12Z)-octadeca-9,12- di.enoate 54

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl stearate 55

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl palmitate 56

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl palmitate 57

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl tetradecanoate 58

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl pentadecanoate 59

CH₂ (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-methylene-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro(3,2-c]isoquinolin-9-yl dodecanoate 60

O (((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a- octahydro-1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9- yl)oxy)methyl tetradecanoate 61 H CH₂ Nalmefene 62 H ONaltrexone

Preparation of Compounds

The compounds used in the reactions described herein are made accordingto organic synthesis techniques known to those skilled in this art,starting from commercially available chemicals and/or from compoundsdescribed in the chemical literature. “Commercially available chemicals”are obtained from standard commercial sources including Acros Organics(Pittsburgh, Pa.), Aldrich Chemical (Milwaukee, Wis., including SigmaChemical and Fluka), Apin Chemicals Ltd. (Milton Park, UK), AvocadoResearch (Lancashire, U.K.), BDH Inc. (Toronto, Canada), Bionet(Cornwall, U.K.), Chemservice Inc. (West Chester, Pa.), CrescentChemical Co. (Hauppauge, N.Y.), Eastman Organic Chemicals, Eastman KodakCompany (Rochester, N.Y.), Fisher Scientific Co. (Pittsburgh, Pa.),Fisons Chemicals (Leicestershire, UK), Frontier Scientific (Logan,Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Key Organics(Cornwall, U.K,), Lancaster Synthesis (Windham, N.H.), MaybridgeChemical Co. Ltd. (Cornwall, U.K.), Parish Chemical Co. (Orem, Utah),Pfaltz & Bauer, Inc. (Waterbury, Conn.), Polyorganix (Houston, Tex.),Pierce Chemical Co. (Rockford, Ill.), Riedel de Haen A G (Hanover,Germany), Spectrum Quality Product, Inc. (New Brunswick, N.J.), TCIAmerica (Portland, Oreg.), Trans World Chemicals, Inc. (Rockville, Md.),and Wako Chemicals USA, Inc. (Richmond, Va.).

Suitable reference books and treatise that detail the synthesis ofreactants useful in the preparation of compounds described herein, orprovide references to articles that describe the preparation, includefor example, “Synthetic Organic Chemistry”, John Wiley & Sons. Inc., NewYork; S, R, Sandler et al., “Organic Functional Group Preparations,” 2ndEd., Academic Press, New York, 1983; H, O. House, “Modern SyntheticReactions”, 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L.Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, NewYork, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanismsand. Structure”, 4th Ed., Wiley-Interscience, New York, 1992. Additionalsuitable reference books and treatise that detail the synthesis ofreactants useful in the preparation of compounds described herein, orprovide references to articles that describe the preparation, includefor example, Fuhrhop, J. and Penzlin G. “Organic Synthesis: Concepts,Methods, Starting Materials”, Second, Revised and Enlarged Edition(1994) John Wiley & Sons ISBN: 3-527-29074-5; Hoffman, R. V. “OrganicChemistry, An Intermediate Text” (1996) Oxford University Press, ISBN0-19-509618-5; Larock, R. C. “Comprehensive Organic Transformations: AGuide to Functional Group Preparations” 2nd Edition (1999) Wiley-VCH,ISBN: 0-471-19031-4; March, J. “Advanced Organic Chemistry: Reactions,Mechanisms, and Structure” 4th Edition (1992) John Wiley & Sons, ISBN:0-471-60180-2; Otera, J. (editor) “Modern Carbonyl Chemistry” (2000)Wiley-VCR ISBN: 3-527-29871-1; Patai, S. “Patai's 1992 Guide to theChemistry of Functional Groups” (1992) Interscience ISBN: 0-471-93022-9;Solomon, T. W. G. “Organic Chemistry” 7th Edition (2000) John Wiley &Sons, ISBN: 0-471-19095-0; Stowell, J. C., “Intermediate OrganicChemistry” 2nd Edition (1993) Wiley-Interscience, ISBN: 0-471-57456-2;“Industrial Organic Chemicals: Starting Materials and Intermediates: AnUllmann's Encyclopedia” (1999) John Wiley & Sons, ISBN: 3-527-29645-X,in 8 volumes; “Organic Reactions” (1942-2000) John Wiley & Sons, in over55 volumes; and “Chemistry of Functional Groups” John Wiley & Sons, in73 volumes.

Specific and analogous reactants are optionally identified through theindices of known chemicals prepared by the Chemical Abstract Service ofthe American Chemical Society, which are available in most public anduniversity libraries, as well as through on-line databases (contact theAmerican Chemical Society, Washington, D.C. for more details). Chemicalsthat are known but not commercially available in catalogs are optionallyprepared by custom chemical synthesis houses, where many of the standardchemical supply houses (e.g., those listed above) provide customsynthesis services. A reference for the preparation and selection ofpharmaceutical salts of the opioid receptor antagonist prodrug compoundsdescribed herein is P. H. Stahl & C. G. Wermuth “Handbook ofPharmaceutical Salts”, Verlag Helvetica Chimica Acta, Zurich, 2002.

Pharmaceutical Compositions

In certain embodiments, the opioid receptor antagonist prodrug compoundas described herein is administered as a pure chemical. In otherembodiments, the opioid receptor antagonist prodrug compound describedherein is combined with a pharmaceutically suitable or acceptablecarrier (also referred to herein as a pharmaceutically suitable (oracceptable) excipient, physiologically suitable (or acceptable)excipient, or physiologically suitable (or acceptable) carrier) selectedon the basis of a chosen route of administration and standardpharmaceutical practice as described, for example, in Remington: TheScience and Practice of Pharmacy (Gennaro, 2E′ Ed. Mack Pub. Co.,Easton, Pa. (2005)),

Provided herein is a pharmaceutical composition comprising at least oneopioid receptor antagonist prodrug compound, or a stereoisomer,pharmaceutically acceptable salt, hydrate, solvate, or N-oxide thereof,together with one or more pharmaceutically acceptable carriers. Thecarrier(s) (or excipient(s)) is acceptable or suitable if the carrier iscompatible with the other ingredients of the composition and notdeleterious to the recipient (i.e., the subject) of the composition.

One embodiment provides a pharmaceutical composition comprising apharmaceutically acceptable excipient and a compound of any one ofFormula (I), (II), (IIa), or (III), or a compound disclosed in Table 1,or a pharmaceutically acceptable salt thereof.

In certain embodiments, the opioid receptor antagonist prodrug compoundas described by any one of Formula (I), (II), (IIa), or (III), or acompound disclosed in Table 1, is substantially pure, in that itcontains less than about 5%, or less than about 1%, or less than about0.1%, of other organic small molecules, such as unreacted intermediatesor synthesis by-products that are created, for example, in one or moreof the steps of a synthesis method.

Suitable oral dosage forms include, for example, tablets, pills,sachets, or capsules of hard or soft gelatin, methylcellulose or ofanother suitable material easily dissolved in the digestive tract. Insome embodiments, suitable nontoxic solid carriers are used whichinclude, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. (See, e.g.,Remington: The Science and Practice of Pharmacy (Gennaro, 21^(st) Ed.Mack Pub. Co., Easton, Pa. (2005)).

In some embodiments, the opioid receptor antagonist prodrug compound asdescribed by any one of Formula (I), (II), (IIa), or (III), or acompound disclosed in Table I, is formulated for administration byinjection. In some instances, the injection formulation is an aqueousformulation. In some instances, the injection formulation is anon-aqueous formulation. In some instances, the injection formulation isan oil-based formulation, such as sesame oil, cottonseed oil, or thelike.

The dose of the composition comprising at least one opioid receptorantagonist prodrug compound as described herein differ, depending uponthe patient's (e.g., human) condition, that is, general health status,age, and other factors.

Pharmaceutical compositions are administered in a manner appropriate tothe disease to be treated (or prevented). An appropriate dose and asuitable duration and frequency of administration will be determined bysuch factors as the condition of the patient, the type and severity ofthe patient's disease, the particular form of the active ingredient, andthe method of administration. In general, an appropriate dose andtreatment regimen provides the composition(s) in an amount sufficient toprovide therapeutic and/or prophylactic benefit (e.g., an improvedclinical outcome, such as more frequent complete or partial remissions,or longer disease-free and/or overall survival, or a lessening ofsymptom severity. Optimal doses are generally determined usingexperimental models and/or clinical trials. The optimal dose dependsupon the body mass, weight, or blood volume of the patient.

Dosing and Therapeutic Regimens

In some embodiments, the pharmaceutical compositions described hereinare administered for therapeutic applications. In some embodiments, thepharmaceutical composition is administered once per day, twice per day,three times per day, four times per day or more. The pharmaceuticalcomposition is administered daily, every day, every alternate day, twodays a week, three days a week, four days a week, five days a week, oncea week, every other week, two weeks per month, three weeks per month,once a month, twice a month, three times per month, or other greater orlesser intervening frequency; also, it could be dosed once every 2months, once every 3 months, once every 4 months, once every 5 months,once every 6 months, once yearly, or with greater or lesser thanaforementioned interval frequency. The pharmaceutical composition isadministered for at least 1 week, 2 weeks, 1 month, 2 months, 3 months,4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months,11 months, 12 months, 18 months, 2 years, 3 years, or more.

In the case wherein the patient's status does not improve, upon thephysician's discretion the administration of the composition is givencontinuously; alternatively, the dose of the composition beingadministered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). In some instances, thelength of the drug holiday varies between 2 days and 1 year, includingby way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days,10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days,100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days,300 days, 320 days, 350 days, 365 days, or 366 days. The dose reductionduring a drug holiday is from 10%400%, including, by way of exampleonly, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be adjusted, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained.

In some embodiments, the amount of given opioid receptor antagonistprodrug compound varies depending upon factors such as the particularcompound, the severity of the disease, the identity (e.g., weight) ofthe subject or host in need. of treatment, but nevertheless is routinelydetermined in a manner known in the art according to the particularcircumstances surrounding the case, including, e.g., the specific agentbeing administered, the route of administration, and the subject or hostbeing treated. In some instances, the desired dose is convenientlypresented in a single dose or as divided doses administeredsimultaneously (or over a short period of time) or at appropriateintervals, for example as two, three, four or more sub-doses per day.

In some embodiments, the amount of given opioid receptor antagonistprodrug compound will typically be in the range of about 0.02 mg toabout 5000 mg per dose. (Note: all prodrug mass quantities are expressedin base moiety equivalents). In some embodiments, the amount of givenopioid receptor antagonist prodrug compound is in the range of about 1mg to about 5000 mg per dose. In some embodiments, the amount of givenopioid receptor antagonist prodrug compound is in the range of about 10mg to about 1600 mg per dose. The desired dose may conveniently bepresented in a single dose or as divided doses administeredsimultaneously (or over a short period of time) or at appropriateintervals, for example as two, three, four or more sub-doses per day.

In some embodiments, the daily dosages appropriate for the opioidreceptor antagonist prodrug compound described herein are from about0.01 mg/kg to about 30 mg/kg. In one embodiment, the daily dosages arefrom about 0.1 mg/kg to about 165 mg/kg. An indicated daily dosage inthe larger mammal, including, but not limited to, humans, is in therange from about 0.5 mg to about 1000 mg, conveniently administered in asingle dose or in divided doses. Suitable unit dosage forms forintramuscular administration include from about 1 to about 5000 mgactive ingredient. In one embodiment, the unit dosage is about 10 mg,about 50 mg, about, 100 mg, about 200 mg, about 500 mg, about 1000 mg,about 2000 mg, about 2500 mg, about 4000 mg, or about 5000 mg.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesmay be altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

Treatment of Behavioral Disorders

In some embodiments, described herein is a method of treating one ormore medical conditions in a subject in need thereof, comprisingadministering to the subject in need thereof an opioid receptorantagonist compound described herein.

In some embodiments, the medical condition is selected from the groupcomprising opioid dependence, alcohol dependence, drug addiction,polydrug addiction and pain.

In some embodiments, described herein is an opioid receptor antagonistcompound for use in reduction of opioid consumption in a patient withopioid dependence.

In some embodiments, described herein is an opioid receptor antagonistcompound for use in reduction of alcohol consumption in a patient withalcohol dependence, pathological gambling shopping addiction or otherdiseases of compulsive behavior.

Provided herein is a method of treating opioid dependence in a patientin need thereof comprising administering a pharmaceutical compositioncomprising a compound of Formula (I), (II), (IIa), or (III), or acompound disclosed in Table 1, or pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable excipient. Provided herein isthe method wherein the pharmaceutical composition is administeredorally. Provided herein is the method wherein the pharmaceuticalcomposition is administered by injection. Provided herein is the methodwherein the pharmaceutical composition is administered by intramuscularinjection. Provided herein is the method wherein the intramuscularinjection is a depot injection. Provided herein is the method whereinthe depot injection provides a therapeutically effective concentrationfor a period of 2 days to 3 months. Provided herein is the methodwherein the depot injection provides a therapeutically effectiveconcentration for a period of about 2 days. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 4 days. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 7 days. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 10 days. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 1 week. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 2 weeks. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 3 weeks. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 4 weeks. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 5 weeks. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 6 weeks. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 1 month. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 2 months. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 3 months. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 4 months. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 5 months. Provided herein is themethod wherein the depot injection provides a therapeutically effectiveconcentration for a period of about 6 months or greater.

Provided herein is a method of treating opioid dependence in a patientin need thereof comprising administering a pharmaceutical compositioncomprising a compound disclosed in Table 3, or pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable excipient.

TABLE 3

R X Chemical Name

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl palmitate

O (4aS,7aS,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl(9Z,12Z)- octadeca-9,12-dienoate

O (4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl palmitoleate

O ((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl myristoleate

O ((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl(Z)- hexadec-6-enoate

O ((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl decanoate

O ((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl undecanoate

O ((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl dodecanoate

O ((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl tridecanoate

O ((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl tetradecanoate

O ((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl pentadecanoate

O ((4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a- hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro- 1H-4,12- methanobenzofuro[3,2-e]isoquinolin-9-yl stearate

Other embodiments and uses will be apparent to one skilled in the art inlight of the present disclosures. The following examples are providedmerely as illustrative of various embodiments and shall not be construedto limit the invention in any way.

EXAMPLES

I. Chemical Synthesis

Unless otherwise noted, reagents and solvents were used as received fromcommercial suppliers. Anhydrous solvents and oven-dried glassware wereused for synthetic transformations sensitive to moisture and/or oxygen.Yields were not optimized. Reaction times are approximate and were notoptimized. Column chromatography and thin layer chromatography (TLC)were performed on silica gel unless otherwise noted. Spectra are givenin ppm (δ) and coupling constants, J are reported in Hertz. For protonspectra the solvent peak was used as the reference peak.

In some embodiments, opioid receptor antagonists prodrug compoundsdisclosed herein are synthesized according to the following examples.

General Scheme 1 for the Synthesis of Nalmefene Prodrugs.

General Scheme 2 for the Synthesis of Naltrexone Prodrugs.

Example 1 Synthesis of(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl((E)-octadec-9-en-1-yl)carbonate

To a mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(8 g, 21.28 mmol, 1 eq, HCl) in H₂O (100 mL) was added K₂CO₃ (8.82 g,63.85 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture wasstirred at 25° C. for 30 min. To a mixture of tetrabutylammonium sulfate(24.73 g, 21.28 mmol, 24.49 mL, 50% solution, 1 eq) in DCM (100 mL) thenthe later mixture was added to the former mixture. Iodomethyl(E)-octadec-9-en-1-yl carbonate (14.44 g, 31.92 mmol, 1.5 eq) obtainedaccording to procedure described in Example 42B, was added and themixture was stirred for 12 hours. The residue was concentrated in vacuumto remove the DCM then was dissolved by saturated solution of NaHCO₃(100 mL). The aqueous phase was extracted with ethyl acetate 600 mL (200mL*3). The combined organic phase was dried with anhydrous Na₂SO₄,filtered and concentrated in vacuum. The residue was purified by silicagel chromatography (Petroleum ether/Ethyl acetate=20/1 to 1/1). Theresidue was further purified by prep-HPLC, MeOH as solvent, selectconventional reverse phase separation as method, separation system isTFA. NaHCO₃ was added to adjust pH to about 8, the aqueous phase wasextracted with ethyl acetate 900 mL(300 mL*3). The combined organicphase was washed with brine (200 mL), dried with anhydrous Na₂Sa₄,filtered and concentrated in vacuum. The compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl[(E)-octadec-9-enyl] carbonate (5 g, 7.46 mmol, 35.03% yield) wasobtained as a yellow oil. M+H⁻=665.5 (LCMS). ¹H NMR (400 MHz, CDCl₃):see FIG. 1.

Example 2 Step 2A: Synthesis of (4-nitrophenyl)undecyl carbonate

To a mixture of undecan-1-ol (40 g, 232.14 mmol, 1 eq) in DCC (600 mL)was added TEA (46.98 g, 464.29 mmol, 64.62 mL, 2 eq) (4-nitrophenyl)carbonochloridate (70.19 g, 348.22 mmol, 1.5 eq) was added to the formermixture portionwise under N₂. The mixture was stirred at 25° C. for 12hr. The reaction mixture was concentrated under reduced pressure toremove solvent. The residue was purified by column chromatography.Compound (4-nitrophenyl) undecyl carbonate (33.95 g, 100.62 mmol, 43.34%yield) was obtained as a yellow solid.

Step 2B: Synthesis of(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylundecyl carbonate

To a mixture of(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(15 g, 39.70 mmol, 1 eq, HCl) in DCM (150 mL) was added TEA (12.05 g,119.09 mmol, 16.58 mL, 3 eq) in one portion at 25° C. under N₂. Themixture was stirred at 25° C. for 30 min, To a mixture of(4-nitrophenyl) undecyl carbonate (26.79 g, 79.39 mmol, 2 eq) in DCM(150 mL), then add to the former mixture, the mixture was stirred at 25°C. for 12 h. The residue was concentrated in vacuum to remove the DCMthen was dissolved by saturated solution of NaHCO₃. The aqueous phasewas extracted with ethyl acetate (200 mL*3). The combined organic phasewas dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum.The residue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=40:1 to 1:1). The residue was further purified byprep-HPLC, MeOH as solvent, select conventional reverse phase separationas method, separation system is TFA, NaHCO₃ was added to adjust pH toabout 8, the aqueous phase was extracted with ethyl acetate (200 mL*3).The combined organic phase was washed with brine (500 mL), dried withanhydrous Na₂SO₄, filtered and concentrated in vacuum. Compound[(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]undecyl carbonate (7.91 g, 14.63 mmol, 36.85% yield) was obtained as ayellow oil. M+H⁺=540.3 (LCMS), ¹H NMR (400 MHz, CDCl₃): see FIG. 2.

Example 3 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylundecyl carbonate

To a mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(15 g, 39.91 mmol, 1 eq, HCl) in DCM (150 mL) was added TEA (12.11 g,119.72 mmol, 16.66 mL, 3 eq) in one portion at 25° C. under N₂. Themixture was stirred at 25° C. for 30 min, To a mixture of(4-nitrophenyl) undecyl carbonate (26.93 g. 79.81 mmol, 2 eq) in DCM(150 mL), then add to the former mixture, the mixture was stirred at 25°C. for 12 h. The mixture was diluted with H₂O (800 mL), extracted withDCM (300 mL*3). The organic phase was washed with brine (300 mL), driedover Na₂SO₄, filtered and the filtrate was concentrated under reducedpressure. The residue was purified by silica gel chromatography(Petroleum ether/Ethyl acetate=40/1 to 1/1). The residue was furtherpurified by prep-HPLC, MeOH as solvent, select conventional reversephase separation as method, separation system is TFA. NaHCO₃ was addedto adjust pH to about 8, the aqueous phase was extracted with ethylacetate (200 mL*3). The combined organic phase was washed with brine(500 mL), dried with anhydrous Na₂SO₄, filtered and concentrated invacuum. The compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]undecyl carbonate (11.40 g, 21.14 mmol, 52.97% yield) was obtained as ayellow oil. M+H⁺=538.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 3.

Example 4 Step 4A: Synthesis of Chloromethyl Undecyl Carbonate

To a mixture of undecan-1-ol (80 g, 464.29 mmol, 1 eq) and pyridine(73.45 g, 928.58 mmol, 74.95 mL, 2 eq) in DCM (600 mL) was addedchloromethyl carbonochloridate (119.73 g, 928.58 mmol, 82.57 mL, 2 eq)dropwise at 0° C. under N₂. The mixture was stirred at 25° C. for 12 h.The reaction mixture was extracted by DCM 1500 mL (500 mL*3). Theorganic phase was separated, washed with brine 30 mL (150 mL*2), driedover Na₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=50/1 to 1:1). Compound chloromethylundecyl carbonate (80 g, 302.13 mmol, 65.07% yield) was obtained as ayellow oil.

Step 4B: Synthesis of Iodomethyl Undecyl Carbonate

To a mixture of chloromethyl undecyl carbonate (30 g, 113.30 mmol, 1 eq)in acetone (400 mL) was added NaHCO₃ (11.42 g, 135.96 mmol, 5.29 mL, 1,2eq) and NaI (20.38 g, 135.96 mmol, 1.2 eq) in one portion at 25° C.under N₂. The mixture was stirred at 25° C. for 12 h in dark. Thereaction mixture was partitioned between EtOAc (400 mL) and H₂O (400mL). The organic phase was separated, washed with brine (80 mL), driedwith anhydrous Na₂SO₄, filtered and concentrated in vacuum. The residuewas purified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=1/0). Compound iodomethyl undecyl carbonate (60 g, 74.33% yield)was obtained as a yellow oil.

Step 4C: Synthesis of(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylundecyl carbonate

To a mixture of(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(8 g, 21.17 mmol, 1 eq, HCl) in H₂O (40 mL) was added K₂CO₃ (8.78 g,63.52 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture wasstirred at 25° C. for 30 min. Then was added tetrabutylammonium sulfate(24.60 g, 21.17 mmol, 24.36 mL, 50% solution, 1 eq) in DCM (10 mL) inone portion at 25° C. Then the mixture was added iodomethyl undecylcarbonate (15.08 g, 42.34 mmol, 2 eq) the mixture was stirred at 25° C.for 11.5 h. The reaction mixture was partitioned between DCM 200 mL(100mL*2) and H₂O 100 mL. The organic phase was separated, washed with brine40 mL, dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum.The residue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=50/1 to 1:1). Then was further purified byprep-HPLC, MeOH as solvent, select conventional reverse phase separationas method, separation system is TFA. NaHCO₃ was added to adjust pH toabout 8, the aqueous phase was extracted with ethyl acetate (400 mL*3).washed with brine 300 mL, dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuum. Compound[(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethylundecyl carbonate (6.9 g) was obtained as a yellow oil. M+H⁺=570.3(LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 4.

Example 5 Synthesis of Example 5:(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylundecyl carbonate

To a mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(8 g, 21.28 mmol, 1 eq, HCl) in H₂O (40 mL) was added K₂CO₃ (8.82 g,63.85 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture wasstirred at 25° C. for 30 min. Then was added tetrabutylammonium sulfate(24.73 g, 21.28 mmol, 24.49 mL, 50% solution, 1 eq) in DCM (40 mL) inone portion at 25° C. Then the mixture was added iodomethyl undecylcarbonate (15.16 g, 42.57 mmol, 2 eq), the mixture was stirred at 25° C.for 11.5 h. The reaction mixture was partitioned between DCM 200 mL(100mL*2) and H₂O 100 mL. The organic phase was separated, washed with brine50 mL, dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum.The residue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=50/1 to 1:1). Then was further purified byprep-HPLC, MeOH as solvent, select conventional reverse phase separationas method, separation system is TFA. NaHCO₃ was added to adjust pH toabout 8, the aqueous phase was extracted with ethyl acetate (400 mL*3),washed with brine 300 mL, dried with anhydrous Na₂SO₄, filtered andconcentrated in vacuum. Compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethylundecyl carbonate (5.9 g) was obtained as a yellow oil. M+H⁺=568.3(LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 5.

Example 6 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldodecyl carbonate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.5 g; ¹H NMR (400 MHz, CDCl₃): seeFIG. 6. Briefly, to a mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(2,5 g, 6.65 mmol, 1 eq, HCl) in DCM (10 mL) was added TEA (2.02 g,19.95 mmol, 2.78 mL, 3 eq) and dodecyl carbonochloridate (2.48 g, 9.98mmol, 1.5 eq). The mixture was stirred at −10° C. for 1 hour and thenwarmed to 25° C. for 4 hours under N₂. The reaction mixture wasconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO2, petroleum ether/ethylacetate=5/1 to 1:1. The compound[(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldodecyl carbonate] was 98.570% pure and obtained as a yellow oil (1.5 g,40.56% yield).

Example 7 Step 7A: Synthesis of chloromethyl(E)-octadec-9-en-1-ylcarbonate

To a mixture of (E)-octadec-9-en-1-ol (22 g, 81.94 mmol, 1 eq) andchloromethyl carbonochloridate (21.13 g, 163.89 mmol, 14.57 mL, 2 eq) inDCM (200 mL) was added pyridine (16.20 g, 204.86 mmol, 16.54 mL, 2.5 eq)dropwise at 0° C. under N₂. The reaction was stirred at 25° C. for 12 hrunder N₂. The reaction mixture was quenched by addition H₂O 400 mL, andextracted with DCM 400 mL*1. The combined organic layers were washedwith brine 300 mL, dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a oil. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0) to giveproduct. Compound chloromethyl (E)-octadec-9-en-1-yl carbonate (50 g,138.52 mmol, 84.52% yield) was obtained as a colorless oil.

Step 7B: Synthesis of iodomethyl(E)-octadec-9-en-1-yl carbonate

To a mixture of chloromethyl (E)-octadec-9-en-1-yl carbonate (30 g,83.11 mmol, 1 eq) and NaI (18.69 g, 124.67 mmol, 1.5 eq) in acetone (300mL) was added NaHCO (8.38 g, 99.73 mmol, 3.88 mL, 1.2 eq) in one portionat 25° C. under N₂. The mixture was stirred at 25° C. for 12 hr. Thereaction mixture was concentrated under reduced pressure to removesolvent. The residue was diluted with 500 mL and extracted with EtOAc800 mL (400 mL*2). The combined organic layers were washed with NaCl aq.400 mL, dried over, filtered and concentrated under reduced pressure togive target product. Compound iodomethyl (E)-octadec-9-en-1-yl carbonate(29 g, 64.10 mmol, 77.13% yield) was obtained as light yellow oil andwas used into the next step without further purification.

Step 7C: Synthesis of(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl((E)-octadec-9-en-1-yl) carbonate

To a mixture of(4R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one;hydrochloride (8 g, 21.17 mmol, 1 eq) and K₂CO₃ (8.78 g, 63.52 mmol, 3eq) in H₂O (200 mL) was stirred at 25° C. for 0.5 hr. Tetrabutylammoniumsulfate (12.30 g, 21.17 mmol, 12.18 mL, 1 eq) DCM (200 mL) was added themixture and stirred for 0.5 hr at 25° C. Iodomethyl(E)-octadec-9-en-1-yl carbonate (14.37 g, 31.76 mmol, 1.5 eq) was addedto the mixture and stirred for 11 hours. The reaction mixture wasconcentrated under reduced pressure to remove DCM. The residue wasdiluted with H₂O (300 mL) and extracted with EtOAc (300 mL*3). Thecombined organic layers were washed with NaCl aq. (300 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The crude was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=10/1 to 3:1) to give target product.Compound[(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl[(E)-octadec-9-enyl] carbonate (8.08 g, 12.09 mmol, 57.12% yield,) wasobtained as a colorless oil. M+F⁺=666.5 (LCMS). ¹H NMR (400 MHz, CDCl₃):see FIG. 7.

Example 8 Synthesis of(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,1a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(E)-octadec-9-enoate

To a mixture of (E)-octadec-9-enoic acid (6.28 g, 22.23 mmol, 1.2 eq) inDCM (100 mL) was added DMF (264.03 mg, 3.61 mmol, 277.93 uL, 0.195 eq)and oxalyl dichloride (8.46 g, 66.69 mmol, 5.84 mL, 3.6 eq) portionwiseat 25° C. under N₂. The mixture was stirred at 25° C. for 30 min, thenconcentrated under reduced pressure. DCM (100 mL) was added in theresidue. To a mixture of(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(7 g, 18.53 mmol, 1 eq, HCl) in DCM (100 mL) was added TEA (3.75 g,37.05 mmol, 5.16 mL, 2 eq), then the former mixture was added in thelater mixture portionwise at 25° C. under N. The mixture was stirred at25° C. for 12 hr. The residue was concentrated in vacuum to remove theDCM then was dissolved by saturated solution of NaHCO₃ (200 mL). Theaqueous phase was extracted with ethyl acetate (100 mL*2). The combinedorganic phase was dried with anhydrous Na₂SO₄, filtered and concentratedin vacuum. The residue was purified by silica gel chromatography(Petroleum ether/Ethyl acetate=40/1 to 1/1). The compound[(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl](E)-octadec-9-enoate (5.36 g, 8.11 mmol, 43.79% yield) was obtained as ayellow oil. M+H⁺=606.2 (LCMS), ¹H NMR (400 MHz, CDCl₃): see FIG. 8.

Example 9 Synthesis of(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl(E)-octadec-9-enoate

To a mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(10 g, 26.60 mmol, 1 eq, HCl) in H₂O (100 mL) was added K₂CO₃ (11.03 g,79.81 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture wasstirred at 25° C. for 30 min. To a mixture of tetrabutylammonium sulfate(30.91 g, 26.60 mmol, 30.61 mL, 50% solution, 1 eq) in DCM (100 mL) thenthe later mixture was added to the former mixture. Iodomethyl(E)-octadec-9-enoate (16.86 g, 39.91 mmol, 1.5 eq), obtained accordingto procedure described in Example 41B, was added and the mixture wasstirred for 12 hours. The mixture was diluted with H₂O (100 mL), collectthe organic phase, then the aqueous phase was extracted with EthylAcetate (300 mL*3), the organic phase was washed with brine (300 mL),dried over Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by silica gel chromatography (Petroleum ether/Ethylacetate=20/1 to 1/1). Then was further purified by prep-HPLC, MeOH assolvent, select conventional reverse phase separation as method,separation system is TFA. NaHCO₃ was added to adjust pH to about 8, theaqueous phase was extracted with ethyl acetate (400 mL*3). The combinedorganic phase was washed with brine (500 mL), dried with anhydrousNa₂SO₄, filtered and concentrated in vacuum. Thecompound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl(E)-octadec-9-enoate (7.16 g, 11.15 mmol, 41.91% yield) was obtained asa yellow oil. M+H⁺=634.4 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 9.

Example 10 Synthesis of(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldecyl carbonate

The title compound was synthesized according to the general Scheme 2 forthe synthesis of naltrexone prodrugs. 1.31 g; ¹H NMR (400 MHz, CDCl₃):see FIG. 10. Briefly, to a mixture of(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(1.9 g, 5.57 mmol, 1 eq) in DCM (15 mL), cooled to −10° C. was added TEA(1.69 g, 16.70 mmol, 2.32 mL, 2 eq) and decyl carbonochloridate (2.46 g,11.13 mmol, 2 eq). The mixture was stirred at 25° C. for 5 hr. Thereaction mixture was concentrated under reduced pressure to give aresidue. The crude product was purified by column chromatography (SiO2,petroleum ether/ethyl acetate=7/3 to 0:1) The compound[(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl decyl carbonate] was97.43% pure and obtained as a yellow oil with a 43.63% yield.

Example 11 Synthesis of(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldodecyl carbonate

The title compound was synthesized according to the general Scheme 2 forthe synthesis of naltrexone prodrugs. 1.5 g; ¹H NMR (400 MHz, CDCl₃):see FIG. 11. Briefly, to a mixture of(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(2.5 g, 6.62 mmol, 1 eq, HCl) and TEA (1.34 g, 13.23 mmol, 1.84 mL, 2eq) in DCM (15 mL) was added dodecyl carbonochloridate (1.56 g, 6.29mmol, 0.95 eq). The mixture was stirred at −10° C. for 1 hr, then warmedto 25° C. and stirred for 4 hr. The reaction mixture was concentratedtinder reduced pressure to give a residue. The residue product waspurified by column chromatography (SiO2, petroleum ether/ethylacetate=7/3 to 0:1). The crude product was purified by prep-HPLC(column: Gemini 200*30 10μ; mobile phase—[water(10 mM NH₄HCO₃)—CAN]; B%70-100%, 12 minutes) The compound[(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldodecyl carbonate] was 99.497% pure and obtained as a white solid (1.5g, 40.74% yield).

Example 12 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylstearate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.2 g; ¹H NMR (400 MHz, CDCl₃): seeFIG. 12.

Example 13 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(Z)-docos-13-enoate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.3 g; ¹H NMR (400 MHz, CDCl₃): seeFIG. 13.

Example 14 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldocosanoate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.5 g; NMR (400 MHz, CDCl₂): seeFIG. 14. Briefly, to a mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(2 g, 5.32 mmol, 1 eq, HCl) in DCM (10 mL) was added TEA (1.62 g, 15.96mmol, 2.22 mL, 3 eq) and docosanoyl chloride (3.82 g, 10.64 mmol, 2 eq)one portion at 25° C. under N₂. The mixture was stirred at 25° C. for 12hr. The reaction mixture was concentrated under reduced pressure to givea residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=5/1 to 1:1) The residue was purified usingprep-HPLC (TFA condition: column: Phenomenx luna (2) C18 250*50 10u;mobile phase: [water(0.1% TFA)-CAN]; B %: 65-95%, 20 minutes]). Thecompound[(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldocosanoate] was 97.01% pure and obtained as a white solid (1.5 g,41.31% yield).

Example 15 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(E)-octadec-9-enoate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.8 g; ¹H NMR (100 MHz, CDCl₃): seeFIG. 15. Briefly, to a mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(2 g, 5.32 mmol, 1 eq, HCl) in DCM (30 mL) was added TEA (1.08 g, 10.64mmol, 1.48 mL, 2 eq) and (E)-octadec-9-enoyl chloride (1.92, 6.38 mmol,1.2 eq). The mixture was stirred at 15° C. for 12 hr. The reactionmixture was mixed with H₂0 (80 mL) and extracted with DCM (80 mL×3). Thecombined organic phase was washed with saturated NaHCO₃ solution (60mL×2) and brine (60 mL×3), dried with anhydrous Na₂SO₄, filtered, andconcentrated in vacuum. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=10/1 to 1:1). Thecompound was purified again using a pre-HPLC column Phenomenex luna C18,250×50mm×10 μm; mobile phase: [water(0.1% TFA)-CAN]; B: 60-90%, 20minutes). After pre-HPLC, the mixture was concentrated under reducedpressure. The aqueous phase was combined with NaHCO₃ to adjust the pH to8, then the aqueous phase was extracted with ethyl acetate (30 mL×4).The combined organic phase was washed with brine (20 mL×1), dried withanhydrous Na₂SO₄, filtered and concentrated in a vacuum The compound[(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(E)-octadec-9-enoate was 95% pure and obtained as a yellow oil (1.8 g,29.57% yield).

Example 16 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylicosanoate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.1 g; ¹H NMR (400 MHz, CDCl₃): seeFIG. 16. Briefly, to a solution of icosanoic acid (5 g, 16.00 mmol, 5.92mL, 1 eq) in DCM (50 mL) was added DMF (116.93 mg, 1.6 mmol, 123.09 μL,0.1 eq), cooled to 0° C., was add (COCl)₂ (2.34 g, 18.40 mmol, 1.61 mL,1.15 eq). TEA (4.86 g, 48.80 mmol, 6.68 mL, 3 eq) and(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(3.01 g, 8.00 mmol, 0.5 eq, HCl). The mixture was stirred at 25° C. for12 hours. The reaction mixture was extracted with H₂O (80 mL×1) and DCM(80 mL×2). The combined organic phase was washed with brine (60 mL×3),dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. Thecompound was purified by column chromatography (SiO₂, petroleumether/ethyl acetate=10/1 to 1:1. The compound[4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylicosanoate] was 100% pure and obtained as a white solid (1.1 g, 10.84%yield).

Example 17 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yloctyl carbonate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs and was obtained as an oil. 1.5 g;¹H NMR (400 MHz, CDCl₃): see FIG. 17.

Example 18 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yldecyl carbonate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs and was obtained as an oil. 1.5 g;¹H NMR (400 MHz, CDCl₃): see FIG. 18.

Example 19 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylhexadecyl carbonate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.8 g; ¹H NMR (400 MHz, CDCl₃): seeFIG. 19. Briefly, to a solution of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(5 g, 13.30 mmol, 1 eq) in DCM (50 mL), cooled to −10° C. TEA (4.04 g,39.91 mmol, 5.55 mL, 3 eq) and hexadecyl carbonochloridate (8.11 g,26.60 mmol, 2 eq) was added. Then, the mixture was stirred at 25° C. for5 hours under N₂ atmosphere. The reaction mixture was extracted with H₂0(80 mL×1) and DCM (80 mL×2). The combined organic phase was washed withbrine (60 mL×3), dried with anhydrous Na₂SO₄, filtered and concentrationin vacuum. The reside and compound was purified by column chromatography(SiO2, petroleum ether/ethyl acetate=10/1 to 1:1) The residue waspurified by prep-HPLC (TFA condition: column—Phenomenex luna C18 250×50mm×10 μm; mobile phase—[water(0.1% TFA)-CAN]; B % 65-95%, 20 minutes).NaHCO₃ was added to adjust pH to 8, and then extracted with EtOAc (20mL×3). The organic layer was evaporated under reduced pressure to getthe final product. The compound[4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofurohexadecyl carbonate] was 99.723% pure and was obtained as a white solid(1.8 g, 12.33% yield).

Example 20 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(9Z,12Z,15Z)-octadeca-9,12,15-trienoate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.4 g; ¹H NMR (400 MHz, CDCl₃): seeFIG. 20.

Example 21 Synthesis of(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylhexadecyl carbonate

The title compound was synthesized according to the general Scheme 2 forthe synthesis of naltrexone prodrugs. 2.15 g; ¹H NMR (400 MHz, CDCl₃):see FIG. 21. Briefly, to a mixture of(3R,4aS,7aR,1.2bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(2 g, 5.29 mmol, 1 eq, HCl0 in DCM (20 mL), cooled to −10° C., was addedTEA (1.61 g, 15.88 mmol, 2.21 mL, 3 eq) and hexadecyl carbonochloridate(3.23 g, 10.59 mmol, 2 eq). Then, the mixture was stirred at 25° C. for5 hours under N atmosphere. The reaction mixture was extracted with H20(80 mL×1) and DCM (80 mL×2). The residue was purified by prep-HPLC (TEAcondition: column Phenomenex luna (2) C18 250×50mm×10 μm; mobilephase—[water(0.1% TEA)-CAN]; B % 60-90%, 20 minutes). NaHCO₃ was addedto adjust pH to 8, and then extracted with EtOAc (20 mL×3). The organiclayer was evaporated under reduced pressure to get the final product.The compound[(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylhexadecyl carbonate] was 97.669% pure and obtained as a white solid(2.15 g, 65.06% yield)

Example 22 Synthesis of(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(Z)-docos-13-enoate

The title compound was synthesized according to the general Scheme 2 forthe synthesis of naltrexone prodrugs. 2.17 g; ¹11 NMR (400 MHz, CDCl₃):see FIG. 22.

Example 23 Synthesis of(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yloctyl carbonate

The title compound was synthesized according to the general Scheme 2 forthe synthesis of naltrexone prodrugs, 1,33 g; ¹H NMR (400 MHz, CDCl₃):see FIG. 23.

Example 24 Step 24A: Synthesis of Chloromethyl Dodecyl Carbonate

To a mixture of dodecan-1-ol (30 g, 161.00 mmol, 1 eq) in DCM (300 mL)was added TEA (32.58 g, 322.00 mmol, 44.82 mL, 2 eq) and chloromethylcarbonochloridate (41.52 g, 322.00 mmol, 28.63 mL, 2 eq) in one portionat 0° C. under N₂. The mixture was heated to 25° C. and stirred for 12hr. The reaction mixture was quenched by addition water 200 mL at 25°C., and then extracted with DCM 100 mL (50 mL*2). The combined organiclayers were dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 80:1).Compound chloromethyl dodecyl carbonate (10.3 g, 36.94 mmol, 22.95%yield) was obtained as a colorless oil.

Step 24B: Synthesis of Iodomethyl Dodecyl Carbonate

To a mixture of chloromethyl dodecyl carbonate (10 g, 35.87 mmol, 1 eq)in acetone (100 mL) was added NaHCO₃ (3.62 g, 43.04 mmol, 1.67 mL, 1.2eq) and NaI (6.45 g, 43.04 mmol, 1.2 eq) in one portion at 25° C. underN₂. The mixture was stirred at 25° C. for 12 hours in dark. The reactionmixture was filtered to remove the insoluble and concentrated underreduced pressure to give a residue. The residue was dissolved in ethylacetate 50 mL and the organic layer was washed with water 60 mL (30mL*2), dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. Compound dodecyl iodomethyl carbonate (12.6g, crude) was obtained as a light red oil. The crude product dodecyliodomethyl carbonate was used into the next step without furtherpurification.

Step 24C: Synthesis of(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyldodecyl carbonate

To a mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-40-diol(4 g, 10.64 mmol, 1 eq, HCl) in H₂O (20 mL) was added K₂CO₃ (4.41 g,31.92 mmol, 3 eq) and the mixture was stirred for 30 min at 20° C.Tetrabutylammonium sulfate (12.37 g, 10.64 mmol, 12.24 mL, 1 eq) and DCM(20 mL) were added to the mixture and the mixture was stirred for 10 minat 20° C. Dodecyl iodomethyl carbonate (9.46 g, 25.54 mmol, 2.4 eq) wasadded to the mixture in one portion at 20° C. under N₂. The mixture wasstirred at 20° C. for 12 hours. The reaction mixture was diluted withwater 20 mL and extracted with DCM 20 mL (10 mL*2). The combined organiclayers were dried, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1:0 to 10:1). Compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyldodecyl carbonate (2.92 g, 5.00 mmol, 47.02% yield) was obtained as acolorless oil. M+H⁺=582.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 24.

Example 25 Step 25A: Synthesis of Chloromethyl Tetradecyl Carbonate

To a mixture of tetradecan-1-ol (30 g, 139.93 mmol, 1 eq) in DCM (300mL) vas added TEA (28.32 g, 279.87 mmol, 38.95 mL, 2 eq) andchloromethyl carbonochloridate (36.09 g, 279.87 mmol, 24.89 mL, 2 eq) inone portion at 0° C. under N₂, then heated to 25° C. for 12 hr. Thereaction mixture was quenched by addition water 200 mL at 25° C., andthen extracted with DCM 100 mL (50 mL*2). The combined organic layerswere dried over Na₂SO₄, filtered and concentrated under reduced pressureto give a residue. The residue was purified by column chromatography(SiO₂, Petroleum ether/Ethyl acetate=1/0 to 80:1). Compound chloromethyltetradecyl carbonate (11 g, 35.85 mmol, 25,62% yield) was obtained as acolorless oil.

Example 25B Synthesis of Iodomethyl Tetradecyl Carbonate

To a mixture of chloromethyl tetradecyl carbonate (11.1 g, 36.17 mmol, 1eq) in acetone (100 mL) was added NaHCO₃ (3.04 g, 36.17 mmol, 1.41 mL, 1eq) and NaI (5.42 g, 36.17 mmol, 1 eq) in one portion at 15° C. underN₂. The mixture was stirred at 15° C. for 12 h. The reaction mixture wasconcentrated under reduced pressure to remove solvent. The residue wasdiluted with ethyl acetate 40 mL and washed with water 40 mL (20 mL*2).The organic layers were dried, filtered and concentrated under reducedpressure to give a residue. The crude product iodomethyl tetradecylcarbonate (13.1 g, 32.89 mmol, 90.92% yield) was obtained as light redoil and used into the next step without further purification.

Step 25C: Synthesis of(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl tetradecyl carbonate

To a mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(3 g, 7.98 mmol, 1 eq, HCl) and iodomethyl tetradecyl carbonate (7.63 g,19.15 mmol, 2.4 eq) in H₂O (30 mL) was added. K₂CO₃ (3.31 g, 23.94 mmol,3 eq) and the mixture was stirred for 0.5 h at 15° C. After 0.5 h,tetrabutylammonium sulfate (4.64 g, 7.98 mmol, 4.59 mL, 1 eq) and DCM(30 mL) were added to the mixture and the mixture was stirred for 10 minat 15° C. After 10 min, iodomethyl tetradecyl carbonate (7.63 g, 19.15mmol, 2.4 eq) was added to the mixture in one portion at 15° C. underN₂. The mixture was stirred at 15° C. for 12 h. The residue was dilutedwith water 10 mL and extracted with DCM 20 mL (10 mL*2). The combinedorganic layers were dried, filtered, and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=1:0 to 10:1).Compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3isoquinoline-9-yl]oxymethyl tetradecyl carbonate (2.0 g, 3.25 mmol,40.76% yield) was obtained as a colorless oil. M+H⁺=610.5 (LCMS). ¹H NMR(400 MHz, CDCl₃): see FIG. 25.

Example 26 Step 26A: Synthesis of chloromethyl(E)-octadec-9-enoate

To a mixture of (E)-octadec-9-enoic acid (2 g, 7.08 mmol, 1 eq) in DCM(15 mL) and H₂O (8 mL) was added NaHCO₃ (2.38 g, 28.32 mmol, 1.10 mL, 4eq) and tetrabutylammonium sulfate (822.29 mg, 708.06 umol, 50%solution, 0.1 eq) in one portion at 25° C. under N₂, then the mixturewas cooled to 0° C. Chloro(chlorosulfonyloxy)methane (1.17 g, 7.08 mmol,1 eq) in DCM (10 mL) was added to the mixture at 0° C. The mixture washeated to 25° C. and stirred for 18 hours. The reaction mixture wasextracted with DCM 30 mL (15 mL*2). The combined organic layers weredried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. Compound chloromethyl (E)-octadec-9-enoate (1.97 g, 5.95mmol, 84.07% yield) was obtained as a white solid and was used into thenext step without purification.

Step 26B: Synthesis of iodomethyl(E)-octadec-9-enoate

To a mixture of chloromethyl (E)-octadec-9-enoate (14.5 g, 43.82 mmol, 1eq) in acetone (140 mL) was added NaHCO₃ (4.42 g, 52.58 mmol, 2.04 mL,1.2 eq) and NaI (7.88 g, 52.58 mmol, 1.2 eq) in one portion at 25° C.under N₂. The mixture was stirred at 25° C. for 12 hours in dark. Thereaction mixture was filtered to remove the insoluble and concentratedunder reduced pressure to give a residue. The residue was dissolved inethyl acetate (100 mL) and the organic layer was washed with brine 100mL, dried over Na₂SO₄, filtered and concentrated under reduced pressureto give a residue. Compound iodomethyl (E)-octadec-9-enoate (18.6 g,crude) was obtained as a brown oil and was used into the next stepwithout purification.

Step 26C: Synthesis of(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyl(E)-octadec-9-enoate

To a mixture of(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(10.09 g, 26.71 mmol, 1 eq, HCl) in H₂O (100 mL) was added K₂CO₃ (11.07g, 80.12 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture wasstirred at 25° C. for 30 min. A mixture of tetrabutylammonium sulfate(15.01 g, 12.92 mmol, 50% solution, 4.84e-1 eq) in DCM (100 mL) then thelater mixture was added to the former mixture. Iodomethyl(E)-octadec-9-enoate (16.92 g, 40.06 mmol, 1.5 eq) was added and themixture was stirred for 12 hours. The mixture was diluted with H₂O (800mL), collect the organic layer, then was extracted with Ethyl Acetate(300 mL*3). All the organic phase was washed with brine (300 mL), driedover Na₂SO₄, filtered and the filtrate was concentrated under reducedpressure. The residue was purified by silica gel chromatography(Petroleum ether/Ethyl acetate=20/1 to 1/1). The residue was furtherpurified by prep-HPLC, MeOH as solvent, select conventional reversephase separation as method, separation system is TFA. NaHCO₃ was addedto adjust pH to about 8, the aqueous phase was extracted with ethylacetate (400 mL*3). The combined organic phase was washed with brine(500 mL), dried with anhydrous Na₂SO₄, filtered and concentrated invacuum. The compound[(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl (E)-octadec-9-enoate(10.20 g, 15.85 mmol, 59.35% yield) was obtained as a yellow oil,M+H⁺=636.4 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 26.

Example 27 Synthesis of(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyltetradecyl carbonate

To a mixture of(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(4.5 g, 11.91 mmol, 1 eq, HCl) in H₂O (30 mL) was added K₂CO₃ (4.94 g,35.73 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture wasstirred at 25° C. for 30 min. tetrabutylammonium sulfate (13.84 g, 11.91mmol, 13.70 mL, 50% solution, 1 eq) and DCM (30 mL) were added to themixture in one portion at 25° C. under N₂. The mixture was stirred at25° C. for 10 min. Iodomethyl tetradecyl carbonate (11.38 g, 28.58 mmol,2.4 eq) was added to the mixture in one portion at 25° C. under N₂. Themixture was stirred at 25° C. for 12 hours. The reaction mixture wasextracted with DCM 30 mL (15 mL*2). The combined organic layers weredried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1/0 to 5:1). Compound[(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyltetradecyl carbonate (2.8 g, 4.53 mmol, 38.05% yield, 99% purity) wasobtained as a colorless oil. ¹H NMR (400 MHz, CDCl₃): see FIG. 27.

Example 28 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylicosyl carbonate

To a mixture of icosyl (4-nitrophenyl) carbonate (9.87 g, 21.28 mmol, 4eq) in DCM (40 mL) was added TEA (538.40 mg, 5.32 mmol, 740.58 uL, 1 eq)and(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(2 g, 5.32 mmol, 1 eq, HCl) in one portion at 15° C. under N₂. Themixture was stirred at 15° C. for 12 hr. The reaction mixture wasextracted with H₂O mL (20 mL*2). The combined organic layers were driedover Na₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by prep-HPLC. Compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]icosyl carbonate (1.6 g, 2.35 mmol, 44.25% yield) was obtained as awhite solid. ¹H NMR (400 MHz, CDCl₃): see FIG. 28.

Example 29 Synthesis of(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyldodecyl carbonate

To a mixture of(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(4.5 g, 11.91 mmol, 1 eq, HCl) in H₂O (30 mL) was added K₂CO₃ (4.94 g,35.73 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture wasstirred at 25° C. for 30 min. tetrabutylammonium sulfate (13.84 g, 11.91mmol, 13.70 mL, 50% solution, 1 eq) and DCM (30 mL) were added to themixture in one portion at 25° C. under N₂. The mixture was stirred at25° C. for 10 min. Dodecyl iodomethyl carbonate (10.58 g, 28.58 mmol,2.4 eq) was added to the mixture in one portion at 25° C. under N2. Themixture was stirred at 25° C. for 12 hours. The reaction mixture wasextracted with DCM 30 mL (15 mL*2). The combined organic layers weredried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate=1/0 to 5:1). Compound[(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyldodecyl carbonate (3.1 g, 5.26 mmol, 44.19% yield, 99.1% purity) wasobtained as a colorless oil. ¹H NMR (400 MHz, CDCl₃): see FIG. 29.

Example 30 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yltridecyl carbonate

To a mixture of (4-nitrophenyl) tridecyl carbonate (5.83 g, 15.96 mmol,2 eq) in DCM (50 mL) was added TEA (2.42 g, 23.94 mmol, 3.33 mL, 3 eq)and(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(3 g, 7.98 mmol, 1 eq, HCl) in one portion at 15° C. under N₂. Themixture was stirred at 15° C. for 12 hr. The reaction mixture wasconcentrated under reduced pressure to remove solvent. The residue waspurified by column chromatography (SiO2, Petroleum ether/Ethylacetate=1/0 to 3:1).[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]tridecyl carbonate (2.3 g, 4.07 mmol, 50.94% yield) was obtained as acolorless oil. ¹H NMR (400 MHz, CDCl₃): see FIG. 30.

Example 31 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yltetradecyl carbonate

To a solution of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(3 g, 7.98 mmol, 1 eq, HCl) in DCM (20 mL) was added TEA (1.62 g, 15.96mmol, 2.22 mL, 2 eq) and tetradecyl carbonochloridate (2.21 g, 7.98mmol, 1 eq). The mixture was stirred at 15° C. for 12 hr. The mixturewas concentrated under reduced pressure. The residue was mixed with H₂O(80 mL) and extracted with DCM (80 mL*3). The combined organic phase waswashed with saturated NaHCO₃ solution (60 mL*2) and brine (60 mL*3),dried with anhydrous Na₂SO₄, filtered and concentrated in vacuum. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=4/1 to 0:1). Compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]tetradecyl carbonate (2 g, 3.41 mmol, 42.79% yield) was obtained as acolorless oil. M+H⁺=580.4 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 31.

Example 32 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-pentadecylcarbonate

To a mixture of (4-nitrophenyl) pentadecyl carbonate (6.28 g, 15.96mmol, 2 eq) in DCM (30 mL) was added TEA (2.42 g, 23.94 mmol, 3.33 mL, 3eq) and[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(3 g, 7.98 mmol, 1 eq, HCl) in one portion at 15° C. under N₂. Themixture was stirred at 15° C. for 12 hr. The residue was purified bycolumn chromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 2:1).CompoundR3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-Apentadecyl carbonate (2.6 g, 1.80 mmol, 22.49% yield) was obtained as awhite solid. M+H⁺=594.3 (LCMS). ¹H NMR (400 MHz, CDCl₃), see FIG. 32.

Example 33 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yloctadecyl carbonate

To a solution of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(2 g, 5.32 mmol, 1 eq, HCl) in DCM (30 mL) was added TEA (1.62 g, 15.96mmol, 2.22 mL, 3 eq) and (4-nitrophenyl) octadecyl carbonate (3.48 g,7.98 mmol, 1.5 eq). The mixture was stirred at 15° C. for 12 hr. Thereaction mixture was concentrated under reduced pressure. The residuewas purified by column chromatography (SiO2, Petroleum ether/Ethylacetate=10/1 to 1:1) and then by pre-HPLC. Compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]octadecyl carbonate (0.8 g, 1.22 mmol, 22.93% yield) was obtained as ayellow oil. M+H⁺=636.5 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 33.

Example 34 Step 34A: Synthesis of Chloromethyl Hexadecyl Carbonate

To a mixture of hexadecan-1-ol (30 g, 123.74 mmol, 1 eq) in DCM (200 mL)was added TEA (25.04 g, 247.48 mmol, 34.45 mL, 2 eq) and chloromethylcarbonochloridate (31.91 g, 247.48 mmol, 22.01 mL, one portion at 0° C.under N₂. The mixture was heated to 20° C. and stirred for 12 hours. Thereaction mixture was quenched by addition water 50 mL at 20° C., andthen extracted with DCM 100 mL (50 mL*2). The combined organic layerswere dried over Na₂SO₄, filtered and concentrated under reduced pressureto give a residue. The residue was purified by column chromatography(SiO₂, Petroleum ether/Ethyl acetate=1/0 to 80:1). Compound chloromethylhexadecyl carbonate (18 g, 53.74 mmol, 43.43% yield) was obtained as awhite solid.

Step 34B: Synthesis of Iodomethyl Hexadecyl Carbonate

To a mixture of chloromethyl hexadecyl carbonate (8 g, 23.89 mmol, 1 eq)in acetone (50 mL) was added NaHCO₃ (2.41 g, 28.66 mmol, 1.11 mL, 1.2eq) and NaI (4.30 g, 28.66 mmol, 1.2 eq) in one portion at 15° C. underN₂. The mixture was stirred at 15° C. for 12 h. The reaction mixture wasconcentrated under reduced pressure to remove solvent. The residue wasdiluted with ethyl acetate 20 mL and washed with water 20 mL (10 mL*2).The organic layers were dried, filtered and concentrated under reducedpressure to give a residue. The crude product hexadecyl iodomethylcarbonate (9 g, crude) was obtained as a light red solid and used intothe next step without further purification. Synthesis of(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylhexadecyl carbonate

To a mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(2.75 g, 7.32 mmol, 1 eq, HCl) and hexadecyl iodomethyl carbonate (7.49g, 17.56 mmol, 2.4 eq) in H₂O (25 mL) was added K₂CO₃ (3.03 g, 21.95mmol, 3 eq) and stirred for 0.5 h at 15° C. After 30 min,tetrabutylammonium sulfate (4.25 g, 7.32 mmol, 4.21 mL, 1 eq) and DCM(25 mL) were added to the mixture and the mixture was stirred for more10 min. After 10 min, hexadecyl iodomethyl carbonate (7.49 g, 17.56mmol, 2.4 eq) was added to the mixture in one portion at 15° C. underN₂. The mixture was stirred at 15° C. for 12 hours. The residue wasdiluted with water 10 ml, and extracted with DCM 20 mL (10 mL*2). Thecombined organic layers were dried, filtered, and concentrated underreduced pressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=1:0 to 10:1).Compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethylhexadecyl carbonate (2.0 g, 3.10 mmol, 42.38% yield) was obtained as acolorless oil. M+H⁺=638.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 34.

Example 35 Synthesis of(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyldecyl carbonate

(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyldecyl carbonate is prepared in a manner analogous to Example 5. To amixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-40-diolin H₂O is added K₂CO₃ in one portion at 25° C. under N₂. The mixture isstirred at 25° C. for 30 min. Then is added tetrabutylammonium sulfatein DCM in one portion at 25° C. Then is added to the reaction mixtureiodomethyl decyl carbonate, the mixture is stirred at 25° C. until thereaction is complete. The reaction mixture is then subjected to workupand the desired product isolated by chromatography as in Example 5. ¹HNMR (400 MHz, CDCl₃): see FIG. 35.

Example 36 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yloleate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.5 g; ¹H NMR (400 MHz, CDCl₃): seeFIG. 36.

Example 37 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2e]isoquinolin-9-yl(9Z,12Z)-octadeca-9,12-dienoate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.3 g; ¹H NMR (100 MHz, CDCl₃): seeFIG. 37.

Example 38 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl3,3-dimethylbutanoate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.2 g; ¹H NMR (400 MHz, CDCl₃): seeFIG. 38.

Example 39 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-ylcyclopentylpropanoate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs. 1.18 g, ¹H NMR (400 MHz, CDCl₃):see FIG. 39.

Example 40 Synthesis of(4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yltert-butylcarbamate

The title compound was synthesized according to the general Scheme 1 forthe synthesis of nalmefene prodrugs and was obtained as a solid. 3.0 g;¹H NMR (400 MHz, CDCl₃): see FIG. 40.

Example 41 Synthesis of(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yloleate

The title compound was synthesized according to the general Scheme 2 forthe synthesis of naltrexone prodrugs. 1.5 g; ¹H NMR (400 MHz, CDCl₃):see FIG. 41.

Example 42 Synthesis of(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl3,3-dimethylbutanoate

The title compound was synthesized according to the general Scheme 2 forthe synthesis of naltrexome prodrugs. 1.3 g; ¹H NMR (400 MHz, CDCl₃):see FIG. 42.

Example 43 Synthesis of(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl3-cyclopentylpropanoate

The title compound was synthesized according to the general Scheme 2 forthe synthesis of naltrexone prodrugs. 1.3 g; ¹H NMR (400 MHz, CDCl₃):see FIG. 43.

Example 44 Step 44A: Synthesis of Chloromethyl Dodecanoate

To a mixture of dodecanoic acid (20 g, 99.84 mmol, 1 eq) in DCM (60 mL)and H₂O (80 mL) was added NaHCO₃ (33.55 g, 399.37 mmol, 15.53 mL, 4 eq)and tetrabutylammonium sulfate (11.60 g, 9.98 mmol, 11.49 mL, 50%purity, 0.1 eq) in one portion at 25° C. under N2, then the mixture wascooled to 0° C. The reactant of chloro(chlorosulfonyloxy)methane (16.47g, 99,84 mmol, 1 eq) in DCM (20 mL) were added to the mixture in oneportion at 0° C. The mixture was heated to 25° C. and stirred for 18hours. The reaction mixture was extracted with DCM 50 mL (25 mL*2). Thecombined organic layers were dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give a residue. The residue waspurified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=1/0 to 80:1). Compound chloromethyl dodecanoate (10.8 g, 43.41mmol, 43.48% yield) was obtained as a colorless oil.

Step 44B: Synthesis of Iodomethyl Dodecanoate

A mixture of chloromethyl dodecanoate (9 g, 36.18 mmol, 1 eq) in acetone(80 mL) was degassed and purged with N₂ for 3 times, and then NaHCO₃(3.04 g, 36.18 mmol, 1.41 mL, 1 eq) and NaI (5.42 g, 36.18 mmol, 1 eq)was added to the mixture in dark, and the result mixture was stirred at15° C. for 12 h under N₂ atmosphere in dark. The reaction mixture wasfiltered and concentrated under reduced pressure to remove solvent. Theresidue was diluted with H₂O O 50 mL, and extracted with EtOAc 120 mL.The combined organic layers were washed with H₂O 100 mL (50 mL*2),dried, filtered and concentrated under reduced pressure to give aresidue. Compound iodomethyl dodecanoate (9 g, crude) was obtained as ayellow liquid and used into the next step without further purification.

Step 44C: Synthesis of(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyldodecanoate

A mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(3.5 g, 10.31 mmol, 1 eq), K₂CO₃ (4.28 g, 30.93 mmol, 3 eq) in H₂O (40mL) was stirred at 15° C. for 30 min and then tetrabutylammonium sulfate(5.99 g, 10.31 mmol, 5.93 mL, 1 eq) and DCM (20 mL) was added to themixture and a solution of iodomethyl dodecanoate (8.42 g, 24.75 mmol,2.4 eq) DCM (20 was added to the mixture and degassed and purged with N₂for 3 times, and then the mixture was stirred at 15° C. for 11.5 h underN₂ atmosphere. The reaction mixture was diluted with H₂O 20 mL andextracted with DCM 20 mL. The combined organic layers were dried,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=1:0 to 20:1). Compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyldodecanoate (1.51 g, 2.70 mmol, 26.14% yield) was obtained as acolorless oil. M+H⁺=552.5 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 44.

Example 45 Step 45A Synthesis of Chloromethyl Tetradecanoate

To a mixture of tetradecanoic acid (20 g, 87.58 mmol, 1 eq) in H₂O (80mL) was added NaHCO₃ (29.43 g. 350.31 mmol, 13.62 mL, 4 eq) andtetrabutylammonium sulfate (10.18 g, 8.76 mmol, 10.08 mL, 50% solution,0.1 eq) and DCM (60 mL) under N₂. The mixture was cooled to 0° C. Thereactant chloro(chlorosulfonyloxy)methane (14.45 g, 87.58 mmol, 1 eq) inDCM (20 mL) was added to the mixture in one portion at 0° C. under N₂.The mixture was heated to 25° C. and stirred for 18 hours. The reactionmixture was extracted with DCM 50 mL (25 mL*2). The combined organiclayers were dried over Na₂SO₄, filtered and concentrated under reducedpressure to give a residue. The residue was purified by columnchromatography (SiO₂, Petroleum ether/Ethyl acetate=1/0 to 80:1).Compound chloromethyl tetradecanoate (15.5 g, 55.99 mmol, 63.93% yield)was obtained as a colorless oil.

Step 45B: Synthesis of Iodomethyl Tetradecanoate

A mixture of chloromethyl tetradecanoate (8 g, 28.90 mmol, 1 eq) inacetone (70 mL) was degassed and purged with N₂ for 3 times, and thenNaHCO₃ (2.43 g, 28.90 mmol, 1.12 mL, 1 eq) and NaI (4.33 g, 28.90 mmol,1 eq) was added to the mixture in dark, the result mixture was stirredat 15° C. for 12 hr under N₂ atmosphere in dark. The reaction mixturewas filtered and concentrated under reduced pressure to remove solvent.The residue was diluted with H₂O 50 mL and extracted with EtOAc 120 mL.The combined organic layers were washed with H₂O 100 mL (50 mL*2), driedover Na₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The crude product was used into the next step without furtherpurification. Compound iodomethyl tetradecanoate (9 g, crude) wasobtained as a yellow solid.

Step 45C: Synthesis of(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyltetradecanoate

A mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-40-diol(3.5 g, 9.31 mmol, 1 eq, HCl), K₂CO₃ (3.86 g, 27.93 mmol, 3 H₂O (30 mL)was stirred at 15° C. for 30 min, and then tetrabutylammonium sulfate(5.41 g, 9.31 mmol, 5.36 mL, 1 eq) and DCM (15 mL) was added to themixture and a solution of iodomethyl tetradecanoate (8.23 g, 22.35 mmol,2.4 eq) in DCM (15 mL) was added to the mixture and degassed and purgedwith N₂ for 3 times, and then the mixture was stirred at 15° C. for 11.5h under N₂ atmosphere. The reaction mixture was diluted with H₂O 20 mLand extracted with DCM 20 mL. The combined organic layers were dried,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=1:0 to 20:1). Compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl tetradecanoate(1.5 g, 2.54 mmol, 27.26% yield) was obtained as a colorless oil.M+H⁺=580.5 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 45.

Example 46 Step 46A: Synthesis of Chloromethyl Hexadecanoate

To a mixture of palmitic acid (20 g, 78.00 mmol, 23.47 mL, 1 eq) in DCM(60 mL) and H₂O (80 mL) was added NaHCO₃ (26.21 g, 311.98 mmol, 12.13mL, 4 eq) and tetrabutylammonium sulfate (9.06 g, 7.80 mmol, 8.97 mL,50% purity, 0.1 eq) in one portion at 25° C. under N₂ and then themixture was cooled to 0° C. The reactant ofchloro(chlorosulfonyloxy)methane (12.87 g, 78.00 mmol, 1 eq) in DCM (20mL) were added to the mixture in one portion at 0° C. The mixture washeated to 25° C. and stirred for 18 hours. The reaction mixture wasextracted with DCM 50 mL (25 mL*2). The combined organic layers weredried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO2,Petroleum ether/Ethyl acetate=1/0 to 80:1). Compound chloromethylhexadecanoate (17.6 g, 57.72 mmol, 71.01% yield) was obtained as a whitesolid.

Step 46B: Synthesis of Iodomethyl Hexadecanoate

A mixture of chloromethyl hexadecanoate (3 g, 9.84 mmol, 1 eq) inacetone (30 mL) was degassed and purged with N₂ for 3 times at 15° C. Indark, and then the mixture was added NaHCO₃ (826.58 mg, 9.84 mmol,382.68 uL, 1 eq) and NaI (1.47 g, 9.84 mmol, 1 eq) and stirred at 15° C.for 12 h under N₂ atmosphere in dark. The reaction mixture filtered andthe filtrate was concentrated under reduced pressure to remove solvent.The residue was diluted with H₂O 20 mL and extracted with EtOAc 60 mL.The combined organic layers were washed with H₂O 40 mL (20 mL*2), dried,filtered and concentrated under reduced pressure to give a residue.Compound iodomethyl hexadecanoate (3.5 g, crude) was obtained as ayellow solid and used into the next step without further purification.

Step 46C: Synthesis of(((4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylhexadecanoate

A mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(1.8 g, 4.79 mmol, 1 eq, HCl), K2CO3 (1.99 g, 14.37 mmol, 3 eq) in H2O(15 mL) was stirred for 30 min, and tetrabutylammonium sulfate (2.78 g,4.79 mmol, 2.75 mL, 1 eq) and DCM (7.5 mL) was added to the mixture, anda solution of iodomethyl hexadecanoate (4.56 g, 11.49 mmol, 2.4 eq) inDCM (7.5 mL) was added to the mixture and degassed and purged with N2for 3 times, and then the mixture was stirred at 15° C. for 11.5 h underN2 atmosphere. The reaction mixture was diluted with H2O 10 mL andextracted with DCM 10 mL. The combined organic layers were dried,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by column chromatography (SiO2, Petroleumether/Ethyl acetate=1:0 to 20:1). Compound[(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethylhexadecanoate (1.6 g, 24.35% yield) was obtained as a colorless oil. M608.6 (LCMS). 1H NMR (400 MHz, CDCl3): see FIG. 46.

Example 47 Synthesis of(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,1a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylhexadecyl carbonate

To a mixture of(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(5.45 g, 14.42 mmol, 1 eq, HCl) in H₂O (30 mL) was added K₂CO₃ (5.98 g,43.27 mmol, 3 eq) in one portion at 25° C. under N₂. The mixture wasstirred at 25° C. for 30 min. tetrabutylammonium sulfate (16.76 g, 14.42mmol, 16.59 mL, 50% solution, 1 eq) and DCM (30 mL) were added to themixture at 25° C. and the mixture was stirred for 10 min at 25° C.hexadecyl iodomethyl carbonate (14.76 g, 34.62 mmol, 2.4 eq) was addedto the mixture in one portion at 25° C. and the mixture was stirred at25° C. for 12 hr. The reaction mixture was extracted with DCM 30 mL (15mL*2). The combined organic layers were dried over Na₂SO₄, filtered andconcentrated under the reduced pressure to give a residue. The residuewas purified by column chromatography (SiO₂, Petroleum ether/Ethylacetate=1/0 to 5:1). Compound[(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethylhexadecyl carbonate (4.68 g, 7.29 mmol, 50.56% yield) was obtained as acolorless oil, M+H⁺=640.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 47.

Example 48 Synthesis of(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methyldodecanoate

A mixture of(3R,4aS,7aS,12bS)-3-(cyclopropylmethyl)-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-4a,9-diol(3.5 g, 10.31 mmol, 1 eq), K₂CO₃ (4.28 g, 30.93 mmol, 3 eq) in H₂O (40mL) was stirred at 15° C. for 30 min and then tetrabutylammonium sulfate(5.99 g, 10.31 mmol, 5.93 mL, 1 eq) and DCM (20 mL) was added to themixture and a solution of iodomethyl dodecanoate (8.42 g, 24.75 mmol,2.4 eq) in DCM (20 mL) was added to the mixture and degassed and purgedwith N₂ for 3 times, and then the mixture was stirred at 15° C. for 11.5h under N₂ atmosphere. The reaction mixture was diluted with H₂O 20 mLand extracted with DCM 20 mL. The combined organic layers were dried,filtered and concentrated under reduced pressure to give a residue. Theresidue was purified by column chromatography (SiO₂, Petroleumether/Ethyl acetate=1:0 to 20:1). Compound [(3R,4aS,7a S,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-methylene-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethyl dodecanoate(1.51 g, 2.70 mmol, 26.14% yield) was obtained as a colorless oil.M+H⁺=554.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 48.

Example 49 Synthesis of(((4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl)oxy)methylhexadecanoate

To a mixture of(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(5 g, 13.23 mmol, 1 eq, HCl) in H₂O (30 mL) was added K₂CO₃ (5.49 g,39.70 mmol, 3 eq) in one portion at 23° C. under N₂. The mixture wasstirred at 25° C. for 30 min. tetrabutylammonium sulfate (15.38 g, 13.23mmol, 15.22 mL, 50% solution, 1 eq) and DCM (30 mL) were added to themixture in one portion at 25° C. under N2. The mixture was stirred at25° C. for 10 min. Iodomethyl hexadecanoate (12.59 g, 31.76 mmol, 2.4eq) was added to the mixture in one portion at 25° C. under N₂. Themixture was stirred at 25° C. for 12 hr. The reaction mixture wasextracted with DCM 30 mL (15 mL*2). The combined organic layers weredried over Na₂SO₄, filtered and concentrated under reduced pressure togive a residue. The residue was purified by column chromatography (SiO₂,Petroleum ether/Ethyl acetate−1/0 to 5:1). Compound[(3R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-9-yl]oxymethylhexadecanoate (5.2 g. 6.14 mmol, 46.40% yield) was obtained as a lightyellow oil. M+H^(|)=610.3 (LCMS). ¹H NMR (400 MHz, CDCl₃): see FIG. 49.

Examples 50-61 Compounds 50-61 Listed in Table 2 can be PreparedAccording to General Scheme 1 for the Synthesis of Nalmefene Prodrugs orGeneral Scheme 2 for the Synthesis of Naltrexone Prodrugs with SuitableStarting Materials

II. Biological Evaluation

Example 1 Plasma and Liver S9 Fraction Stability Assay

Plasma stability determination of the test compounds in rat, dog,cynomolgus monkey and human plasma is performed using HPLC-MS. For rat,incubations are carried out in 96-well polypropylene plates in 5aliquots of 70 μL each (one for each time point). Test compounds (10 μM,final solvent concentration 1%) are incubated at 37° C. Five time pointsare analyzed (0, 15, 120, 480 and 1440 min). For dog, monkey and human,test compounds (2 μM, final solvent concentration 1%) were alsoincubated at 37° C. and analyzed at five time points (0, 10, 30, 60 and120 min). All incubations are performed in duplicates. The samples areanalyzed by HPLC-MS. The percentage of parent compound remaining afterincubation in plasma is determined. Nalmefene dodecanoate and nalmefenepalmitate were previously reported (Gaekens et al, Journal of ControlledRelease 232 (2016) 196-202). Results are provided in Table 4a-d.

TABLE 4a Rat Plasma Stability % remaining at time point in minutesCompound 0 min 15 min 120 min 480 min 1440 min 36 100 74.4 66.3 39.819.1  6 100 31.3 7.6 4 1.6 53 100 46.85 19.6 7.3 1.9 11 100 0.7 0.3 0.20 54 100 98.8 62.4 45.6 19.4 14 100 94 112 107 120 15 100 72 69.3 52 3517 100 0 0 0 0 18 100 0.6 0.3 0.1 0 19 100 70.1 71.5 43.8 33.3 55 10077.3 47.2 27.1 15.6 23 100 0 0 0 0 10 100 0 0 0 0 21 100 73.5 68.3 59.840.9 50 100 114.5 102.7 83.3 73.9 51 100 107.4 105.3 88.2 92.7  8 10044.2 34.3 15.3 5.2 57 100 10.5 3.2 1.2 0 58 100 37 20 2.9 1.8 30 10065.7 40.3 30.5 22.2 31 100 123.2 73.5 54.4 46.4 24 100 32 11.8 8.2 3 25100 82.6 69.3 62.2 51.7 59 100 5.9 0.7 0.2 0 56 100 73.1 56.7 35.8 16.8Aripiprazole 100 84 85.5 76.4 46 Lauroxil (plasma cleavage control)Enalapril maleate 100 44 2.9 8.2 4.8 salt (plasma cleavage control)

TABLE 4b Dog Plasma stability % remaining at time point in minutesCompound 0 min 15 min 120 min 480 min 1440 min  6 100 89.1 96.9 107.2101.2 15 100 82.5 80.6 90.1 86.1 24 100 94.7 104.5 115.7 116.2Aripiprazole lauroxil 100 103 81.3 78.6 74.3 Paliperidone palmitate 10091.2 88.4 95.9 93.1

TABLE 4c Monkey Plasma stability % remaining at time point in minutesCompound 0 min 15 min 120 min 480 min 1440 min  6 100 88.7 74.3 68.470.1 15 100 74.5 72.7 80.6 76.5 24 100 90.7 93.5 96.2 97.8 Aripiprazolelauroxil 100 95.2 94.5 75.5 83.2 Paliperidone palmitate 100 92 89.5 98.492.5

TABLE 4d Human Plasma stability % remaining at time point in minutesCompound 0 min 15 min 120 min 480 min 1440 min  6 100 97.6 102 109.8115.1 15 100 83.3 82.5 107.1 109.0 24 100 94 102.3 105.3 112.5Aripiprazole lauroxil 100 99.7 80 73.2 75.5 Paliperidone palmitate 100103.7 97.6 109.1 104.1

Liver S9 fraction stability determination of the test compounds in dog,cynomolgus monkey and human is performed using HPLC-MS. Test compound (2μM, 0.1% DMSO, 1% Methanol final concentration) was assessed forstability in a 50 μl phosphate buffer containing 1.0 mg/ml S9 proteinfrom each of the three species and 5 mM D-saccharic acid-1,4-lactone.Samples were incubated at 37° C. for 60 minutes and the % compoundremaining was assessed.

TABLE 4e Liver S9 Fraction Stability % remaining at 60 minutes CompoundDog Monkey Human  6 17.1 0 5.3 15 80.9 76.7 59.8 24 3.6 3 3.5Aripiprazole lauroxil 66.1 56.6 47.8 Paliperidone 56.4 57.4 47.8palmitate

Example 2 Opioid Receptor Binding Assay

Receptor binding assays were performed to assess the ability ofcompounds to inhibit binding to radiolabeled ligand. First, the IC50values were determined for select compounds for all 3 opioid receptorsubtypes (DOR, MOR and KOR) and compared these values to that of theparent molecule, Nalmefene. The general observation is that prodrugderivatization greatly reduces the binding affinity to the opioidreceptors. In some cases by several orders of magnitude.

Apparatus

Unifilter-96 GF/C filter plates, Perkin Elmer (Cat #6005174)

96 well conical polypropylene plates, Agilent (Cat #5042-385)

TopSeal—A sealing film, Perkin Elmer (Cat #6005250)

TopCount NXT HTS, (Perkin Elmer)

MicroBeta² (Perkin Elmer)

Cell harvest C961961, (Perkin Elmer)

Reagents

The stable cell lines were established and prepared cell membraneobtained using these cell lines.

³H-diprenophrine (PerkinElmer, Cat: NET1121250UC, Lot: 2143599)

³H-DAMGO (PerkinElmer, Cat: NET9022501UC, Lot; 2139100)

³H-DADLE (PerkinElmer, Cat: NET648250UC, Lot: 2060549)

Tris base (Sigma, Cat: T6066-1KG), prepare 1M stock and adjust pH to7.4.

0.5M EDTA (Invitrogen, Cat: 15575-038)

1M MgCl₂ (Sigma, Cat: M1028-100 ml)

PEI (Poly ethyleneimine) (Sigma, Cat: P3143)

Microscint 20 cocktail (Perkin Elmer, Cat: 6013329)

Naltrindole (Sigma, Cat; N115)

(±)trans-U-50488 (Sigma, Cat; D8040)

DAMGO (Sigma, Cat: E7384)

Assay Buffer

Op-delta Assay Buffer Final Concentration Tris- 50 mM HCl MgCl2 10 mMEDTA  1 mM Adjust pH to 7.4, stored at 4° C.

Op-kappa Assay Buffer Final Concentration Tris- 50 mM HCl Adjust pH to7.4, stored at 4° C.

Op-mu Assay Buffer Final Concentration Tris- 50 mM HCl MgCl2  5 mMAdjust pH to 7.4, stored at 4° C.

Wash Buffer

op-kappa, op-delta and op-mu Wash Buffer Final Concentration Tris- 50 mMHCl Adjust pH to 7.4, stored at 4° C.

Methods

1) Membrane and Radio Ligand Preparation

Membrane Concentration Radio ligand Target (ug/well) Radio ligandconcentraton (nM) DOR 6.7 [3H]-DADLE 0.5 MOR 20 [3H]DAMGO 0.5 KOR 6.7[3H]Diprenorphine 0.3

2) Compound Preparation

Ref: starting Compound Final Starting conc. In Ref: Final Starting NSBcompound starting conc. In Conc. In assay source plate Conc. In assayConc. in assay Target source plate (mM) plate (nM) (mM) plate (nM) plateDOR 2 10000 0.02 100 naltrindole (1 μM) MOR 2 10000 0.2 1000 naltrindole(1 μM) KOR 2 10000 0.2 1000 trans-U-50488 (5 μM)

3) Assay Procedure

1) Transfer 1 μl of specified concentration compound to assay plateaccording to the plate map for nonspecific binding. Transfer 1 μl ofDMSO to assay plate according to plate map for total binding.

2) Follow the plate map. Dispense 99 μl of membrane stocks into theplate.

3) Add 100 μl of radio ligand.

4) Seal the plates. Incubate at RT for 1 hour.

5) Soak the Unifilter-96 GE/C filter plates with 50 ul of 0.3% PEI perwell for at least 0.5 hour at room temperature.

6) When binding assays are completed, filter the reaction mixturethrough GF/C plates using Perkin Elmer Filtermate Harvester, and thenwash each plate for 4 times with cold wash buffer.

7) Dry the filter plates for 1 hour at 50 degrees.

8) After drying, seal the bottom of the filter plate wells using PerkinElmer Unifilter-96 backing seal tape. Add 50 μl of Perkin ElmerMicroscint 20 cocktail.

Seal top of filter plates with Perkin Elmer TopSeal—A sealing film.

9) Count ³H trapped on filter using Perkin Elmer MicroBeta2 Readersecond day.

10) Analyze the data with GraphPad Prism 5. Calculate the “Inhibition[%Control]” using the equation: % Inh=(1−Background subtracted Assayvalue/Background subtracted HC value)*100.

Results

Reference KOR U-50488 DOR naltrindole MOR DAMGO

TABLE 5a DOR MaxDose Compound IC50 (nM) (nM) % Inh@MaxDose 30 1854 1000084.3 32 >10000 10000 43.2 25 9540 10000 62  6 499.5 10000 70.9 23 106.710000 95.6 24 2121 10000 48 34 >10000 10000 22.7 44 190.1 10000 79.1 4568.4 10000 73.8 46 1360 10000 72.3 nalmefene 18.1 1000 94.5 Naltrindole0.2 100 98.8

TABLE 5b KOR MaxDose Compound IC50 (nM) (nM) % Inh@MaxDose 30 88.7 1000097.6 32 2116 10000 80.1 25 889.3 10000 76.9  6 51.1 10000 99.6 23 11.710000 101.6 24 37.5 10000 82.9 34 1767 10000 68.1 44 18.4 10000 99.2 455.5 10000 100.1 46 33.85 10000 101.8 nalmefene 2 1000 103 U-50488 10.11000 101.6

TABLE 5c MOR MaxDose Compound IC50 (nM) (nM) % Inh@MaxDose 30 9 1000082.5 32 17.4 10000 75.9 25 5 10000 87.3  6 14.9 10000 102.5 23 2.8 1000098.7 34 12.55 10000 92.7 44 3.029 10000 83.1 45 2 10000 93.7 46 4.52310000 102.3 24 22.4 10000 88.7 nalmefene 0.4 1000 103.5 DAMGO 1.4 100099.3

Example 3 Solubility Determination

A known amount of test substance (˜40 mg) was weighed into the vial, 100μL of oil was added and heated to 60° C. and then system was slurried toreach equilibrium. More oil was added until clear solution was obtainedor the solubility was <50 mg/mL. Then the clear solution was placed atroom temperature (25° C.) for 24 h to confirm whether there was solidprecipitation. Extra oil was added into the vial once compoundprecipitated out and then the system was re-equilibrated at 1000 rpm atroom temperature (25° C.). Final concentration was determined by HPLCmethod as described below in Table 6a and 6b.

TABLE 6a Reagent Name Grade Company Lot No. Purified Water HPLCWuXiAppTec N/A Cottonseed oil N/A SIGMA 038K000G Cottonseed oil SUPERREFINED CRODA 1070292 Sesame oil HPLC CRODA 1115393 Ethanol HPLC J. T.Baker 155943 ACN HPLC Merck I0904530 735

TABLE 6b Instrument Name Model Company Serial number Water Milli-QDirect 8 MILLIPORE PDS-PF-WPE-01 Purification Equipment BalanceMettler-Toledo Mettler-Toledo PDS-PF-BAL-08 XPR10 Stirrer C-MAG MS 10IKA PDS-PF-ST-01 Balance Mettler-Toledo Mettler-Toledo PDS-PF-BAL-03 MX5HPLC Shimadzu 20AB Shimadzu PDS-PF-HPLC-12 Thermomixer YQH-0623Eppendorf PDS-PF-TM-02

The HPLC method for Compounds 6, 12-20, and 36-43 is provided in Table7.

TABLE 7 HPLC Method 1 Instrument Shimadzu 20AB HPLC Column AscentisExpress C18, 10 cm*4.6 mm, 2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100%ACN Flow Rate 1.0 ml/min Inject volume 10 μL Analysis Time 20 min ColumnTemp. 40° C. Wavelength 280 nm Gradient Time Program (min) A % B % 0 8020 9 10 90 9.01 10 90 14 80 20 20 80 20 20.01 Stop

The HPLC method for Compounds 10, 21-23, 53, 55, 56, nalmefene, andnaltrexone is provided in Table 8.

TABLE 8 HPLC Method 2 Instrument Shimadzu 20AB HPLC Column AscentisExpress C18, 10 cm*4.6 mm, 2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100%ACN Flow Rate 1.0 ml/min Inject volume 10 uL Analysis Time 13 min ColumnTemp. 40° C. Wavelength 284 nm Gradient Time Program (min) A % B % 0 955 9 10 90 11 10 90 11.01 95 5 13 95 5 13.01 Stop

The HPLC method for Compounds 3-5, 8, 24-25, 26-34, 44-51, 54, 55, 57,59, and 60 is provided in Table 9.

TABLE 9 HPLC Method 3 Instrument Shimadzu 20AB HPLC Column AscentisExpress C18, 10 cm*4.6 mm, 2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100%ACN Flow Rate 1.0 ml/min Inject volume 10 μL Analysis Time 13 min ColumnTemp. 40° C. Wavelength 280 nm Gradient Time Program (min) A % B % 0 9010 4 35 65 25 20 80 27 10 90 27.01 90 10 30 90 10 30.01 Stop

TABLE 10 Equilibrium Approximate Solubility(mg/mL) in Excipientsolubility (mg/mL) Heating Temp. Obs. Temp. Compound Castor oilCottonseed oil Sesame oil Ethanol Water (° C.) (° C.)36 >217 >418.7 >411.6 20-30  0.005 40 40 36 N/A >407   >408   N/A N/A 6025 37 >226 >405.3 >408.4 75-90  0.002 40 40 37 N/A 68.5-82   80-102 N/AN/A 60 25 38 >248 >406.9 230-411 160-200  0.617 40 40 38 N/A N/A N/A N/A0.21 60 40 39 >218  225-402.2 >400.3 150-200  0.018 40 40 39 N/A 104-139106-141 N/A N/A 60 25  6 N/A >420.8 >402.8 120-170  0.002 40 40  6N/A >397   >386   N/A N/A 60 25 41 N/A >407   >403   >201  0.084 40 4041 N/A <50  <50  N/A N/A 60 25 53 N/A >403.8 >406.3 >404  0.0143 60 4053 N/A >408.5 >401.9 N/A N/A 60 25 42 N/A <51  <52  78-93  0.245 40 4042 N/A <26   <25.8 N/A  0.068 60 40 43 N/A <50  <50  >257 0.05 40 40 43N/A 140-210 133-199 N/A N/A 60 40 43 (in water) N/A N/A N/A N/A  0.04840 40 11 N/A >407   >407   >218  0.0005 40 40 11 N/A <51  <50  N/A N/A60 25 11 after slurry in Heptane N/A N/A N/A N/A  0.0026 N/A N/A 12 N/A100-120 70-95 65-75  0.043 40 40 12 N/A >398.5 >411.1 N/A N/A 60 40 12N/A <51  <50  N/A N/A 60 25 12(in water) N/A N/A N/A N/A  0.026 40 40 54N/A <51  <50  N/A N/A 60 25 54 (in water) N/A N/A N/A N/A <0.001 40 4013 N/A >381   >406   >214  0.002 40 40 13 N/A <50   91-121 N/A N/A 60 2514 N/A <50  <51  69-83 0.01 40 40 14 N/A <25  <25  N/A N/A 60 40 14(inwater) N/A N/A N/A N/A  0.0056 40 40 14 after slurry in Heptane N/A N/AN/A N/A  <0.0005 N/A N/A 14 after slurry in EtOAc N/A N/A N/A N/A <0.0005 N/A N/A 15 N/A <51  <52   <20  <0.0002 40 40 15N/A >401   >404   N/A N/A 60 40 15 N/A >402.6 >396.5 N/A N/A 60 25 15N/A N/A  >403.94 N/A N/A 60 25 16 N/A 57-67 68-81  <25  0.2059 40 40 16N/A 201-403 202-404 N/A  0.011 60 40 17 N/A >416   >416   >209  0.000840 40 17 N/A >408   >487   N/A N/A 60 25 18 N/A >403   >403   >209 <0.0002 40 40 18 N/A >400   >402   N/A N/A 60 25 19 N/A 67-80 103-13727-42  0.0468 40 40 20 N/A >414   >406   157-197  <0.0002 40 40 20 N/A68-82  80-100 N/A N/A 60 25 61 (Nalmefene) N/A  <25.0  <25.5 102-137 0.025 60 40 62 (Naltrexone) N/A  <26.6  <25.9 103-137  0.295 60 40 56N/A 25-50 25-50   <40.4  0.0679 60 40 56 N/A <50  <50  N/A N/A 60 25 55N/A >400.1 >394.8   >401.1  0.0066 60 40 55 N/A <50  <50  N/A N/A 60 2555 (after ETOAc slurry) N/A N/A N/A N/A  <0.0005 N/A N/A 22N/A >405.9 >401.9   >397.7  0.0314 60 40 22 N/A 67-80 67-80 N/A N/A 6025 23 N/A >411.8 >420.7   >419.3  0.0094 60 40 23 N/A >407.7 >423.9 N/AN/A 60 25 10 N/A >420.7 >404.1   >404.7  0.0006 60 40 10 N/A 100-134<51  N/A N/A 60 25 21 N/A  101-134.8  101-134.5  50-100  0.0006 60 40 50N/A <51  <52  N/A N/A 60 25 50 (in water) N/A N/A N/A N/A <0.001 40 4051 N/A <50  <46  N/A N/A 60 25 51 (in water) N/A N/A N/A N/A <0.001 4040  8* N/A <50  <52  N/A N/A 60 25  8 (in water)* N/A N/A N/A N/A 0.5740 40 56 NMF palmitate N/A 25-50 25-50   <40.4  0.0066 60 40 56 NMFpalmitate N/A <50  <50  N/A N/A 60 25 57 N/A >423.7 >417.3 N/A N/A 60 2557 (in water) N/A N/A N/A N/A <0.001 40 40 58 N/A 101.8-203.6 99.4-198.8 N/A N/A 60 25 58 (in water) N/A N/A N/A N/A  0.0014 40 40 59NMF dodecanoate N/A >414   >393.3 N/A N/A 60 25 59 NMF dodecanoate (inN/A N/A N/A N/A <0.001 40 40 water) 30 N/A  252.5-404.06 252.55-398.6 N/A N/A 60 25 30 (in water) N/A N/A N/A N/A <0.001 40 40 31N/A >416.9 >413.5 N/A N/A 60 25 31 (in water) N/A N/A N/A N/A <0.001 4040 32 N/A >395.8 >410   N/A N/A 60 25 32(in water) N/A N/A N/A N/A 0.0328 40 40 33 N/A <50  <51  N/A N/A 60 25 33(in water) N/A N/A N/AN/A <0.001 40 40 28 N/A  <50.2  <50.6 N/A N/A 60 25 28(in water) N/A N/AN/A N/A <0.001 40 40 24 N/A >403   338-368 N/A N/A 60 25 24(in water)N/A N/A N/A N/A <0.001 40 40 25 N/A >414   >404   N/A N/A 60 25 25(inwater) N/A N/A N/A N/A <0.001 40 40 34 N/A >404   >405   N/A N/A 60 2534 (in water) N/A N/A N/A N/A <0.001 40 40 44 N/A >407   208-231 N/A N/A60 25 44 (in water) N/A N/A N/A N/A <0.001 40 40 45 N/A >402   267-288N/A N/A 60 25 45 (in water) N/A N/A N/A N/A <0.001 40 40 46 N/A >395.5202-269 N/A N/A 60 25 46 (in water) N/A N/A N/A N/A <0.001 40 40 29N/A >410.2 >416.5 N/A N/A 60 25 29 (in water) N/A N/A N/A N/A <0.001 4040 27 N/A >394.7 >399.2 N/A N/A 60 25 27 (in water) N/A N/A N/A N/A<0.001 40 40 47 N/A >405.5 >404.9 N/A N/A 60 25 47 (in water) N/A N/AN/A N/A <0.001 40 40 48 N/A >410.5 >404.8 N/A N/A 60 25 48 (in water)N/A N/A N/A N/A  0.003 40 40 26 N/A 204.3-239  <354   N/A N/A 60 25 26(in water) N/A N/A N/A N/A <0.001 40 40 49 N/A <50  <51  N/A N/A 60 2549 (in water) N/A N/A N/A N/A  0.028 40 40 Equilibrium Heating Temp.Obs. Temp. Solubility by volumetric flask method solubility (mg/mL) (°C.) (° C.) 8** N/A N/A >399.8 N/A N/A 60 25 8 (in water)** N/A N/A N/AN/A <0.001 40 40 3 N/A  >399.12 >400.7 N/A N/A 60 25 3 (in water) N/AN/A N/A N/A <0.001 40 40 4 N/A >395.3 >400.4 N/A N/A 60 25 4 (in water)N/A N/A N/A N/A <0.001 40 40 5 N/A >401.8 >400.6 N/A N/A 60 25 5 (inwater) N/A N/A N/A N/A <0.001 40 40 *Data from compound 8 initial failedbatch due to non-optimized synthesis **Data from compound 8 second batchafter successfully optimized synthesis

Example 4 Stability Determination of Drug Product

Compounds were resuspended in oil vehicles, stored at room temperaturefor the indicated time period and assessed by HPLC. Data is presented asabsolute percentage loss normalized to 30 days. Nalmefene dodecanoatewere previously reported (Gaekens et al, Journal of Controlled Release232 (2016) 196-202).

TABLE 11 Reagents Name Grade Company Lot No. Water HPLC WuXiAppTec N/AACN HPLC Merck I0911030735 Cottonseed oil N/A SIGMA 038K000G Cottonseedoil SUPER REFINED CRODA 1070292 Sesame oil N/A ACROS A0377665 Sesame oilHPLC CRODA 1115393 TFA HPLC J&K LN20M33 THF HPLC MACRON 1613729801

TABLE 12 Instruments Name Model Company Serial number Water Milli-QDirect 8 MILLIPORE PDS-PF-WPE-01 Purification Equipment BalanceMettler-Toledo Mettler-Toledo PDS-PF-BAL-05 XP6 HPLC Shimadzu 20ABShimadzu PDS-PF-HPLC-12

TABLE 13 HPLC Method Instrument Shimadzu 20AB HPLC Column AscentisExpress C18, 10 cm*4.6 mm, 2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100%ACN Flow Rate 1.2 ml/min Inject volume 10 uL Analysis Time 30 min ColumnTemp. 40° C. Wavelength 280 nm Diluent THF Gradient Time Program (min) A% B % 0 90 10 4 35 65 25 20 80 27 10 90 27.01 90 10 30 90 10 30.01 Stop

TABLE 14 % Avg Purity % Avg Purity Degradation in Cottonseed OilDegradation in Sesame Oil Compound (normalized 30 day) (normalized 30day) 29 0.13 0.32 27 0.13 0.32 32 — 0.13 24 0.43 (0.01) 25 0.28 (0.11)31 1.15 0.08 6 1.48 0.31 15 0.86 0.10 17 1.05 0.26 18 1.25 0.71 23 0.350.09 5 0.40 0.28 59 4.60 0.51 34 (0.11) 0.02 44 0.36 (0.08) 45 0.31 0.0946 0.04 Not tested 47 0.43 Not tested 48 0.60 Not tested 3 0.04 0.07

Example 4 Stability Determination of Drug Substance

Compounds were stored at room temperature for the indicated time periodand assessed by HPLC. Data is presented as absolute percentage lossnormalized to 30 days. Nalmefene dodecanoate were previously reported(Gaekens et al, Journal of Controlled Release 232 (2016) 196-202).

TABLE 15 Reagent Name Grade Company Lot No. Purified Water HPLCWuXiAppTec N/A ACN HPLC Merck JA056730 TFA HPLC J&K LN20M33 THF HPLCMACRON 1613729801

TABLE 16 Instrument Name Model Company Serial number Water Milli-QDirect 8 MILLIPORE PDS-PF-WPE-01 Purification Equipment BalanceMettler-Toledo Mettler-Toledo PDS-PF-BAL-08 XPR10 Balance Mettler-ToledoMettler-Toledo PDS-PF-BAL-03 MX5 HPLC Shimadzu 20AB ShimadzuPDS-PF-HPLC-12

TABLE 17 HPLC method for compounds 10, 11, 21, 36, and 53 HPLC Method 1Instrument Shimadzu 20AB HPLC Column Ascentis Express C18, 10 cm*4.6 mm,2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100% ACN Flow Rate 1.2 ml/minInject volume 10 uL Analysis Time 32 min Column Temp. 40° C. DiluentACN:H2O(3:1) Wavelength 280 nm Gradient Time Program (min) A % B % 0 9010 4 40 60 25 5 95 27 5 95 27.01 90 10 32 90 10 32.01 Stop

TABLE 18 HPLC method for compounds 14, 19 and 55 HPLC Method 2Instrument Shimadzu 20AB HPLC Column Ascentis Express C18, 10 cm*4.6 mm,2.7 μm Gradient A = 0.1% TFA/H₂O, B = 100% ACN Flow Rate 1.2 ml/minInject volume 10 uL Analysis Time 32 min Column Temp. 40° C. DiluentACN:H2O(3:1) Wavelength 280 nm Gradient Time Program (min) A % B % 0 9010 4 35 65 25 20 80 27 10 90 27.01 90 10 30 90 10 30 Stop

TABLE 19 HPLC method for compounds 1, 3-9, 15, 17, 18, 23-27, 29, 31,32, 34, 35, 44-48, 57, and 59 HPLC Method 3 Instrument Shimadzu 20ABHPLC Column Ascentis Express C18, 10 cm*4.6 mm, 2.7 μm Gradient A = 0.1%TFA/H₂O, B = 100% ACN Flow Rate 1.2 ml/min Inject volume 10 uL AnalysisTime 32 min Column Temp. 40° C. Diluent THF Wavelength 280 nm GradientTime Program (min) A % B % 0 90 10 4 35 65 25 20 80 27 10 90 27.01 90 1030 90 10 30 Stop

TABLE 20 Purity (after room % Degradation Purity temperature NormalizedCompound (initial) storage) % Degradation to 30 days 36 97.70% 81.19%16.51% 5.27% 6 99.00% 99.56% −0.56% −0.18% 53 97.00% 89.90% 7.10% 3.95%11 98.20% 98.96% −0.76% −0.28% 14 97.01% 97.90% −0.89% −0.33% 15 98.40%98.64% −0.24% −0.10% 17 96.30% 98.38% −2.08% −0.84% 18 98.44% 98.43%0.01% 0.00% 19 99.70% 100.00% −0.30% −0.13% 55 98.80% 98.89% −0.09%−0.04% 23 97.30% 99.97% −2.67% −1.31% 10 97.40% 80.74% 16.66% 8.19% 2197.60% 98.81% −1.21% −0.60% 57 99.60% 99.84% −0.24% −0.17% 59 97.41%99.09% −1.68% −1.23% 31 98.80% 98.80% 0.00% 0.00% 32 99.00% 99.22%−0.22% −0.23% 24 99.00% 98.86% 0.14% 0.11% 25 99.00% 98.33% 0.67% 0.38%34 98.00% 97.87% 0.13% 0.07% 44 98.00% 98.40% −0.40% −0.22% 45 98.00%98.49% −0.49% −0.28% 46 97.00% 96.97% 0.03% 0.02% 29 99.00% 99.43%−0.43% −0.46% 27 98.90% 98.11% 0.79% 0.85% 47 99.00% 99.24% −0.24%−0.29% 48 99.00% 98.89% 0.11% 0.15% 26 99.00% 95.89% 3.11% 0.72% 799.00% 99.32% −0.32% −0.10% 8 98.30% 98.48% −0.18% −0.05% 9 99.00%97.59% 1.41% 0.35% 1 99.00% 99.18% −0.18% −0.07% 3 99.00% 99.62% −0.62%−0.30% 4 99.00% 99.73% −0.73% −0.24% 5 100.00% 100.00% 0.00% 0.00% 35100.00% 100.00% 0.00% 0.00%

Example 5 Physical Characterization of Solid State Drug Substance

The analysis of the physical characteristics of drug substances thatwere in a solid state was conducted using polarized light microscopy(PLM), X-ray powder diffractometer (XRPD) assessment, DifferentialScanning Calorimetry (DSC) and Thermal Gravimetric Analysis (TGA). ForPLM, samples were dispersed in immersion oil and were observed using anocular lens (10×) and objective lens (20×) under crossed polarizers. ForXRPD, samples were run on a diffractometer using the following method:Tube—Cu: K-alpha(λ=1.54179 {acute over (Å)}); Generator—Voltage 40 kV,Current 40 mA; Scan scope—3 to 40°; sample rotation speed—15 rpm;scanning rate—10 deg/min. For DSC, ˜1 mg of sample was tested using acrimped aluminum pan and covered by a lid with a hole, heated from roomtemperature to 300° C. at a speed of 10° C./minute. For TGA, 2-5 mg ofsample was placed in an open platinum pan and heated from roomtemperature to 300° C. at a rate of 10° C./minute. Nalmefene palmitatewere previously reported (Gaekens et al, Journal of Controlled Release232 (2016) 196-202).

TABLE 21 XRPD, TGA and DSC results X-ray Powder Thermal GravimetricDifferential scanning Cpd # Diffraction Analysis calorimetry 11 CrystalForm with obvious Weight loss of 0.30% at Single endothermic peakbirefringence 120° C. following at 56.71° C. which could bedecomposition melting point. 12 Crystal Form with obvious Weight loss of0.19% at Single endothermic peak birefringence 120° C. following at51.94° C. which could be decomposition melting point. 14 Weakcrystallinity of the Weight loss of 1.649% at Single endothermic peakcompound 120° C. following at 124.66° C. which could decomposition bemelting point. 16 Crystal Form with obvious Weight loss of 0.403% atSingle endothermic peak birefringence 120° C. following at 59.04° C.which could be decomposition melting point. 19 Crystal Form with obviousWeight loss of 0.1623% at Single endothermic peak birefringence 120° C.following at 56.31° C. which could be decomposition melting point. 56Crystal Form with obvious Weight loss of 0.1815% at Two endothermic peakat birefringence 120° C. following 48.95° C. and 59.75° C. decomposition55 Crystal Form with obvious Weight loss of 0.2597% at Singleendothermic peak birefringence 120° C. following at 48.24° C. whichcould be decomposition melting point. 10 Crystal Form with obviousWeight loss of 0.3047% at Single endothermic peak birefringence 120° C.following at 47.18° C. which could be decomposition melting point. 40Crystal form with partial Weight loss of 0.57% at Two endothermic peakat birefringence 120° C. following 107.6° C. and 195.0° C. decomposition21 Crystal Form with partial Weight loss of 1.133% at Single endothermicpeak birefringence. 120° C. following at 70.84° C. which could bedecomposition melting point. 31 Crystal Form with obvious Weight loss of0.7244% at Single endothermic peak birefringence 120° C. following at45.15° C. which could be decomposition melting point.

Example 5 Polymorph Screening of Solid State Drug Substances

In order to identify stable polymorph forms of solid state drugsubstances, approximately 50 mg of compound (nalmefene or naltrexoneequivalents) was weighed into vials. Next, 500 μL of the indicatedsolvents was added and the suspension was stirred at 700 rpm, 50° C. for72 hours. For samples in suspension, solids were separated by centrifuge(10 minutes, 14000 rpm) and dried in vacuum oven at 30° C. overnight.For samples in solution, solids were generated by evaporation (stir barremoved and covered with aluminum foil with pinholes, then dried invacuum oven at 30° C. overnight). Dried solids were characterized byXRPD, TGA and DSC. Results are presented in Table 23.

TABLE 23 Target XRPD Compound conc. Visual observation results #Solvents (mg/ml) RT 50° C. Dry method and appearance (Dried) Comment 11Heptane 100 Clear Clear Evaporation/white powder Pattern B Initialcrystal form (pattern A) likely MTBE Clear Clear Evaporation/whitepowder Pattern A to be stable form; Pattern A generated IsopropanolSlurry Clear Evaporation/white powder Pattern A by evaporation withMTBE, IPA, EtOAc Slurry Clear Evaporation/white powder Pattern A EtOAcand Acetone, Compound Acetone Slurry Clear Evaporation/white powderPattern A evaporated in heptane was different form 14 Heptane 100 SlurrySlurry Centrifugation/white powder Pattern B Initial crystal form(pattern A) MTBE Clear Clear Evaporation/white powder Pattern A appearsunstable; Same crystal from Isopropanol Clear Clear Evaporation/whitepowder Pattern A generated by evaporation from EtOAc Slurry ClearEvaporation/white powder Pattern A MTBE, IPA, EtOAc and acetone andAcetone Slimy Clear Evaporation/white powder Pattern A might beunstable. Form evaporated in heptane different form and likely morestable 16 Heptane 100 Slurry Slurry Centrifugation/white powder PatternA Initial crystal form (pattern A) is MTBE Clear Clear Evaporation/whitepowder Pattern A likely stable form. Same crystal form IsopropanolSlurry Slurry Centrifugation/white powder Pattern A (pattern A) wasgenerated by EtOAc Slurry Clear Evaporation/white powder Pattern Aevaporation from MTBE, EtOAc and Acetone Slurry Clear Evaporation/whitepowder Pattern A Acetone. Same crystal form (pattern A) generated byslurry in Heptane and IPA 19 Heptane 100 Slurry Clear Evaporation/whitepowder Pattern A Initial crystal form (pattern A) likely MTBE ClearClear Evaporation/white powder Pattern A to be stable fomr. Same crystalform Isopropanol Slurry Clear Evaporation/white powder Pattern A(pattern A) was generated by EtOAc Slurry Clear Evaporation/white powderPattern A evaporation from Heptane, MTBE, Acetone Slurry ClearEvaporation/white powder Pattern A IPA, EtOAc and Acetone 55 Heptane 100Slurry Clear Evaporation/white powder Pattern B Cannot determine whichcrystal form MTBE Clear Clear Evaporation/white powder Pattern B is morestable. Same form (pattern B) Isopropanol Slurry Clear Evaporation/whitepowder Pattern B was generated by evaporation from EtOAc Slurry ClearEvaporation/white powder Pattern B Heptane, MTBE, IPA, EtOAc and AcetoneSlurry Clear Evaporation/white powder Pattern B Acetone. 10 Heptane 100Slurry Slurry Centrifugation/White wax Pattern A Initial crystal form(pattern A) is a MTBE Clear Clear Evaporation/White wax Pattern A stableform. Same form (pattern A) Isopropanol Slurry Clear Evaporation/Whitewax Pattern A generated by evaporation from EtOAc Slurry ClearEvaporation/White wax Pattern A MTBE, IPA, EtOAc and Acetone. AcetoneSlurry Clear Evaporation/White wax Pattern A Pattern A also generated byslurry in heptane. 21 Heptane 100 Slurry Clear Evaporation/white powderPattern A Initial crystal form (pattern A) is a MTBE Clear ClearEvaporation/white powder Pattern A stable form. Same crystal formIsopropanol Slurry Clear Evaporation/white powder Pattern A (pattern A)was generated by EtOAc Slurry Clear Evaporation/white powder Pattern Aevaporation from Heptane, MTBE, Acetone Slurry Clear Evaporation/whitepowder Pattern A IPA, EtOAc and Acetone 31 Heptane 100 Slurry ClearEvaporation/white powder Pattern A Initial crystal form (pattern A) veryMTBE Slurry Clear Evaporation/white powder Pattern A likely to be astable form. Same Isopropanol Clear Clear Evaporation/white powderPattern A crystal form (pattern A) was generated EtOAc Clear ClearEvaporation/white powder Pattern A by evaporation from MTBE, IPA,Acetone Slurry Clear Evaporation/white powder Pattern A EtOAc andacetone 40 Heptane 100 Slurry Slurry Centrifugation/white powder PatternA Initial crystal form (pattern A) is a MTBE Clear ClearEvaporation/white powder Amorphous stable form. Same crystal formIsopropanol Slurry Clear Evaporation/oil Amorphous (pattern A) wasgenerated by slurry in EtOAc Slurry Clear Evaporation/white powderPattern A heptane and evaporation form EtOAc. Acetone Slurry ClearEvaporation/white powder Amorphous Compound became amorphous byevaporation from MTBE, IPA and acetone.

III. Preparation of Pharmaceutical Dosage Forms

Example 1 Oral Capsule

The active ingredient is a compound of Table 1, or a pharmaceuticallyacceptable salt thereof. A capsule for oral administration is preparedby mixing 1-1000 mg of active ingredient with starch or other suitablepowder blend. The mixture is incorporated into an oral dosage unit suchas a hard gelatin capsule, which is suitable for oral administration.

Example 2 Solution for Injection

The active ingredient is a compound of Table 1, or a pharmaceuticallyacceptable salt thereof, and is formulated as a solution in sesame oil,cottonseed oil, castor oil or other pharmaceutically acceptablelipophilic excipient, preferably at a concentration of greater than 100mg/mL. The resulting solution is administered by intramuscularinjection.

Compounds were resuspended to 1 mL at the indicated concentrations (inmg/ml base equivalents) by mixing with magnetic stirring (1000 rpm) at60° C. until a homogeneous clear solution was achieved, then cooled downto room temperature and stored protected from light. Appearance of oilformulations was observed and recorded at room temperature (25° C.) atinitial, 2 hours, and 24 hours. Samples for “Assay” measurements weretaken at initial, 2 hours and 24 hours post resuspension and subjectedto HPLC analysis where actual concentration was based on a standardcurve (Assay=Concentration(measured by HPLC)/Actual concentration(byweight)×100%). Purity was calculated at indicated time points based onthe percentage of area under the curve of the main peak from the HPLCspectrogram. Syringability was assessed by drawing through a 21 Gaugeneedle. Some indicated samples were assessed for Appearance, Assay andPurity after 7 months in 40° C./75% Relative Humidity. Data arepresented in Table 21.

TABLE 24 Target Conc. (mg/ml) Appearance Assay Compound Vehicle Volumein Base eq. Initial 2 hours 24 hours Initial 2 hours 6 Sesame 1 mL 200Light Light Light 102.26% 102.32% oil + 10 amber amber amber uL Benzyltransparent transparent transparent Alcohol oil oil oil Sesame 1 mL 300Light Light Light 99.82% 99.66% oil + 10 amber amber amber uLBenzyltransparent transparent transparent Alcohol oil oil oil Sesame 1 mL 400Light Light Light 98.06% 97.94% oil + 10 amber amber amber uLBenzyltransparent transparent transparent Alcohol oil oil oil 15 Sesame 1 mL200 Light Light Light 107.68% 109.70% oil + 10 amber amber amberuLBenzyl transparent transparent transparent Alcohol oil oil oil Sesame1 mL 300 Light Light Light 101.55% 108.28% oil + 10 amber amber amberuLBenzyl transparent transparent transparent Alcohol oil oil oil Sesame1 mL 400 Light Light Light 93.52% 99.92% oil + 10 amber amber amberuLBenzyl transparent transparent transparent Alcohol oil oil oil 17Sesame 1 mL 200 Light Light Light 98.80% 98.66% oil + 10 amber amberamber uLBenzyl transparent transparent transparent Alcohol oil oil oilSesame 1 mL 300 Light Light Light 105.43% 105.79% oil + 10 amber amberamber uLBenzyl transparent transparent transparent Alcohol oil oil oilSesame 1 mL 400 Light Light Light 98.51% 98.82% oil + 10 amber amberamber uLBenzyl transparent transparent transparent Alcohol oil oil oil18 Sesame 1 mL 200 Light Light Light 97.56% 97.79% oil + 10 amber amberamber uLBenzyl transparent transparent transparent Alcohol oil oil oilSesame 1 mL 300 Light Light Light 99.00% 100.43% oil + 10 amber amberamber uLBenzyl transparent transparent transparent Alcohol oil oil oilSesame 1 mL 400 Light Light Light 98.54% 98.70% oil + 10 amber amberamber uLBenzyl transparent transparent transparent Alcohol oil oil oil23 Sesame 1 mL 200 Light Light Light 97.22% 97.54% oil + 10 amber amberamber uLBenzyl transparent transparent transparent Alcohol oil oil oilSesame 1 mL 300 Light Light Light 100.34% 100.25% oil + 10 amber amberamber uLBenzyl transparent transparent transparent Alcohol oil oil oilSesame 1 mL 400 Light Light Light 97.54% 97.92% oil + 10 amber amberamber uLBenzyl transparent transparent transparent Alcohol oil oil oil57 Sesame 1 mL 200 Light Light Light 104.75% 104.82% oil + 10 amberamber amber uLBenzyl transparent transparent transparent Alcohol oil oiloil Sesame 1 mL 300 Light Light Light 102.67% 102.47% oil + 10 amberamber amber uLBenzyl transparent transparent transparent Alcohol oil oiloil Sesame 1 mL 400 Light Light Light 101.03% 101.18% oil + 10 amberamber amber uLBenzyl transparent transparent transparent Alcohol oil oiloil 31 Sesame 1 mL 200 Light Light Light 101.90% 100.35% oil + 10 amberamber amber uLBenzyl transparent transparent transparent Alcohol oil oiloil Sesame 1 mL 300 Light Light White 105.84% 95.68% oil + 10 amberamber wax uLBenzyl transparent transparent Alcohol oil oil Sesame 1 mL400 Light Light White 99.16% 95.81% oil + 10 amber amber wax uLBenzyltransparent transparent Alcohol oil oil 32 Sesame 1 mL 300 Light LightLight 95.61% 96.59% oil + 10 amber amber amber uLBenzyl transparenttransparent transparent Alcohol oil oil oil Sesame 1 mL 350 Light LightWhite 102.17% 103.30% oil + 10 amber amber wax uLBenzyl transparenttransparent Alcohol oil oil Sesame 1 mL 400 Light Light White 97.21%97.10% oil + 10 amber amber wax uLBenzyl transparent transparent Alcoholoil oil 24 Cottonssed 1 mL 400 Light Light Light 107.62% 99.58% oil + 10clear clear clear uLBenzyl yellow oil yellow oil yellow oil Alcohol 29Sesame 1 mL 400 Light Light Light 103.07% 104.83% oil + 10 amber amberamber uLBenzyl transparent transparent transparent Alcohol oil oil oil27 Sesame 1 mL 400 Light Light Light 95.52% 98.43% oil + 10 amber amberamber uLBenzyl transparent transparent transparent Alcohol oil oil oil47 Sesame 1 mL 400 Light Light Light 102.41% 101.60% oil + 10 amberamber amber uLBenzyl transparent transparent transparent Alcohol oil oiloil Follow up observation Assay Purity Syringability (21G needle) atroom Compound Vehicle 24 hours Initial 2 hours 24 hours Initial 2 hours24 hours temperature 6 Sesame 99.30% 99.48% 99.46% 99.37% feasiblefeasible feasible Remains in oil + 10 solution uL Benzyl Alcohol Sesame102.90% 99.37% 99.35% 99.44% feasible feasible feasible oil + 10uLBenzyl Alcohol Sesame 98.43% 99.37% 99.39% 99.37% feasible feasiblefeasible oil + 10 uLBenzyl Alcohol 15 Sesame 109.70% 96.38% 96.44%96.34% feasible feasible feasible Remains in oil + 10 solution uLBenzylAlcohol Sesame 103.00% 96.44% 96.38% 96.45% feasible feasible feasibleoil + 10 uLBenzyl Alcohol Sesame 94.15% 96.42% 96.28% 96.41% feasiblefeasible feasible oil + 10 uLBenzyl Alcohol 17 Sesame 98.29% 98.61%98.64% 98.61% feasible feasible feasible Remains in oil + 10 solutionuLBenzyl Alcohol Sesame 105.65% 98.61% 98.60% 98.63% feasible feasiblefeasible oil + 10 uLBenzyl Alcohol Sesame 97.58% 98.63% 98.60% 98.59%feasible feasible feasible oil + 10 uLBenzyl Alcohol 18 Sesame 97.85%99.15% 99.05% 99.07% feasible feasible feasible Remains in oil + 10solution uLBenzyl Alcohol Sesame 99.52% 98.93% 99.06% 98.84% feasiblefeasible feasible oil + 10 uLBenzyl Alcohol Sesame 98.77% 99.17% 99.13%99.14% feasible feasible feasible oil + 10 uLBenzyl Alcohol 23 Sesame97.10% 99.50% 99.48% 99.47% feasible feasible feasible Remains in oil +10 solution uLBenzyl Alcohol Sesame 99.51% 99.49% 99.48% 99.50% feasiblefeasible feasible oil + 10 uLBenzyl Alcohol Sesame 95.81% 99.51% 99.48%99.49% feasible feasible feasible oil + 10 uLBenzyl Alcohol 57 Sesame104.02% 99.55% 99.49% 99.54% feasible feasible feasible Insoluble: oil +10 gross uLBenzyl precipitant/ Alcohol phase Sesame 100.66% 99.69%99.67% 99.65% feasible feasible feasible seperation oil + 10 after 23days uLBenzyl Alcohol Sesame 100.34% 99.63% 99.59% 99.63% feasiblefeasible feasible oil + 10 uLBenzyl Alcohol 31 Sesame N/A 99.29% 99.28%N/A feasible feasible feasible Insoluble: oil + 10 gross uLBenzylprecipitant/ Alcohol phase Sesame N/A 99.14% 99.31% N/A feasiblefeasible N/A seperation oil + 10 after 24 hr uLBenzyl Alcohol Sesame N/A99.23% 99.25% N/A feasible feasible N/A oil + 10 uLBenzyl Alcohol 32Sesame 95.42% 98.78% 98.63% 98.78% feasible feasible feasible Insoluble:oil + 10 gross uLBenzyl precipitant/ Alcohol phase Sesame N/A 98.79%98.76% N/A feasible feasible N/A seperation oil + 10 after 24 hruLBenzyl Alcohol Sesame N/A 98.80% 98.78% N/A feasible feasible N/Aoil + 10 uLBenzyl Alcohol 24 Cottonssed 100.58% 99.02% 98.86% 98.93%feasible feasible feasible Remains in oil + 10 solution uLBenzyl Alcohol29 Sesame 103.35% 99.21% 99.21% 99.28% feasible feasible feasible Drugoil + 10 substance uLBenzyl shown to be Alcohol unstable in oil solutionat 4 months 40 C./75% RH 27 Sesame 96.46% 99.33% 99.30% 99.09% feasiblefeasible feasible Insoluble: oil + 10 gross uLBenzyl precipitant/Alcohol phase seperation after 90 days 47 Sesame 101.72% 99.37% 99.31%99.26% feasible feasible feasible Insoluble: oil + 10 gross uLBenzylprecipitant/ Alcohol phase seperation after 24 hr

TABLE 25A Target conc. (mg/ml) Appearance Sample Nalmefene 7 months inCompound # Vehicle Volume amount Initial 2 hours 24 hours 40 C./75% RH25 Sesame 1 + 1 mL 400 Light Light Light Light hazy oil + 20 uL amberamber amber oil, possible Benzyl transparent transparent transparentimmiscible Alcohol oil oil oil particles. 34 Sesame 1 + 1 mL 400 LightLight Light Yellow oil + 20 uL amber amber amber hazy oil, Benzyltransparent transparent transparent possible Alcohol oil oil oilimmiscible oil droplets. 44 J&K 1 mL 350 Light Light Light Cottonseedclear clear clear oil + 10 uL yellow oil yellow oil yellow oil BenzylAlcohol 45 J&K 1 + 1 mL 300 Light Light Light Yellow Cottonseed yellowyellow yellow hazy oil, oil + 20 uL clear oil clear oil clear oilpossible Benzyl small oil Alcohol droplets 46 J&K 1 + 1 mL 300 LightLight Light Yellow Cottonseed yellow yellow yellow hazy non- oil + 20 uLhazy oil hazy oil hazy oil homogenous Benzyl oil with Alcohol obviousimmiscible oil droplets 48 Sesame 1 mL 400 Light Light Light oil + 10 uLhazy oil hazy oil hazy oil Benzyl Alcohol Assay 7 months in 7 months in7 months in 40 C./75% 40 C./75% 40 C./75% Compound # Vehicle Initial 2hours 24 hours RH-1 RH-2 RH-3 25 Sesame 102.48% 102.90% 108.2.5% 98.02%94.38% 98.31% oil + 20 uL Benzyl Alcohol 34 Sesame 101.77% 102.07%101.11% 102.01% 99.07% 97.99% oil + 20 uL Benzyl Alcohol 44 J&K 101.41%102.44% 102.32% Cottonseed oil + 10 uL Benzyl Alcohol 45 J&K 98.86%98.68% 98.64% 100.57% 99.54% 98.78% Cottonseed oil + 20 uL BenzylAlcohol 46 J&K 98.94% 99.28% 99.00% 106.24% 108.43% 107.85% Cottonseedoil + 20 uL Benzyl Alcohol 48 Sesame 97.50% 96.63% 96.39% oil + 10 uLBenzyl Alcohol

TABLE 25B Purity 7 months 7 months 7 months Syringeability Follow-up inin in (21 Gauge needle) Follow up observation at 7 Com- 40 C./75% 40C./75% 40 C./75% 24 observation at months at 40 C./ pound # Initial 2hours 24 hours RH-1 RH-2 RH-3 Initial 2 hours hours room temp 75% RH 2598.36% 98.64% 98.57% 97.07% 97.13% 97.07% feasible feasible feasibleRemains in solution No clearly visible particles on PLM microcopy 3498.59% 98.58% 98.50% 99.35% 99.44% 99.46% feasible feasible feasibleRemains in solution No clearly visible particles on PLM microcopy 4498.68% 98.41% 98.73% feasible feasible feasible Insoluble: grossly hazywith visible solid precipitate after 20 days 45 99.58% 101.17% 100.62%94.60% 94.68% 94.67% feasible feasible feasible Remains in solutionPossible small visible particles on PLM microcopy 46 97.96% 97.96%97.95% 95.48% 95.42% 95.54% feasible feasible feasible Insoluble:grossly hazy Obvious with visible solid particles on on precipitateafter PLM 20 days microscopy 48 98.97% 98.80% 98.53% feasible feasiblefeasible Insoluble: grossly hazy with visible solid precipitate after170 days. Crystalline particles on PLM microscopy. Drug substancechanged from colorless oil to white wax within 4 weeks.

TABLE 26A Target conc. (mg/ml) Appearance Sample Nalmefene 3 months in 6months Compound # Vehicle Volume amount Initial 24 hours 40 C./75% RH in40 C./75% RH 6 Sesame oil + 50 uL 5 mL 400 Light amber Light amber Lightamber Light amber Benzyl Alcohol transparent oil transparent oiltransparent oil (at transparent oil 25 C.) 15 Sesame oil + 50 uL 5 mL400 Light amber Light amber Light amber Light amber Benzyl Alcoholtransparent oil transparent oil transparent oil transparent oil 17Sesame oil + 50 uL 5 mL 400 Light amber Light amber Light amber Lightamber Benzyl Alcohol transparent oil transparent oil transparent oiltransparent oil 18 Sesame oil + 50 uL 5 mL 400 Light amber Light amberLight amber Light amber Benzyl Alcohol transparent oil transparent oiltransparent oil transparent oil 9 Sesame oil + 50 uL 5 mL 400 Lightamber Light amber Light amber Light amber Benzyl Alcohol transparent oiltransparent oil transparent oil transparent oil 59 Sesame oil + 10 uL 5mL 86 Light amber Light amber Benzyl Alcohol transparent oil transparentoil 24 Cottonseed oil + 200 20 mL 400 Pale yellow oil uL Benzyl Alcohol45 Sesame oil + 1% 20 mL 400 clear oil Benzyl alcohol Sesame oil + 1% 1mL 400 clear oil Benzyl alcohol Sesame oil + 1% 1 mL 400 clear oilBenzyl alcohol Sesame oil + 1% 1 mL 400 clear oil Benzyl alcohol 34Sesame oil + 100 10 mL 400 Brown oil ul Benzyl Alcohol 7 Sesame oil + 1%5 + 1 + 1 + 1 mL 400 Almost clear Benzyl alcohol solution 43 Sesameoil + 1% 10 mL 400 Brown oil Benzyl alcohol 9 Sesame oil + 1% 5 ml 400Almost clear Benzyl alcohol 1 ml solution 1 ml 1 ml 1 Sesame oil + 1%5 + 1 + 1 mL 400 Slightly turbid Benzyl alcohol oil 3 Sesame oil + 1%5 + 5 mL 400 Light amber Light amber Benzyl alcohol transparent oiltransparent oil (6 hr) 4 Sesame oil + 1% 5 + 1 + 1 mL 400 Almost clearBenzyl alcohol solution 5 Sesame oil + 1% 1 + 1 + 1 mL 400 Light amberBenzyl alcohol transparent oil 35 Sesame oil + 1% 10 mL 400 Almost clearBenzyl alcohol oil

TABLE 26B Assay 3 months in 40 C./75% Compound # Initial-1 Initial-2Initial-3 24 hr-1 24 hr-2 24 hr-3 RH-1 6 103.19% 106.47% 104.72% 100.81%100.60% 101.40% 102.15% (25 C.) 15 99.37% 98.69% 101.17% 97.06% 101.32%99.43%  97.75% 17 98.84% 99.62% 99.49% 98.02% 98.22% 98.07% 101.42% 1893.67% 98.55% 96.11% 95.75% 95.77% 93.03% 102.46% 9 102.27% 100.89%101.56% 99.70% 97.28% 90.59% 101.74% 59 102.52% 102.59% 102.71% 103.05%102.47% 102.90% 24 112.73% 105.24% 103.76% 45 98.12% 98.99% 100.30%100.05% 101.02% 99.92% 99.86% 101.14% 99.39% 99.91% 99.03% 99.99% 3496.78% 97.68% 98.16% 7 99.73% 100.16% 101.37% 43 99.27% 99.39% 97.86% 999.23% 99.79% 99.83% 99.35% 98.76% 98.66% 98.93% 100.93% 103.17% 109.00%109.82% 111.59% 1 110.88% 111.97% 110.59% 3 N/A N/A N/A 97.91% 97.55%94.13% (6 days) (6 (6 days) days) 4 100.31% 97.12% 97.52% 5 96.64%95.72% 96.29% 35 98.21% 96.79% 99.37% Assay 3 months 3 months 6 months 6months 6 months in in in in in 40 C./75% 40 C./75% 40 C./75% 40 C./75%40 C./75% Compound # RH-2 RH-3 RH-1 RH-2 RH-3  6 101.9% 99.07% 101.25%101.08% 101.24% (25 C.) (25 C.) 15  97.98%  97.53% 101.91% 101.82%102.09% 17 101.39% 101.30% 103.37% 103.24% 103.18% 18 103.29% 102.94%99.73% 100.49% 100.23%  9 102.03% 101.57% 98.23% 98.80% 93.52% 59 24 4534  7 43  9  1  3  4  5 35

TABLE 26C Purity 3 months 3 months 3 months 6 months 6 months 6 monthsSyringeability in in in in in in (21 Gauge needle) Follow up 40 C./75%40 C./75% 40 C./75% 40 C./75% 40 C./75% 40 C./75% 24 3 6 observation atCompound # Initial-1 Initial-2 Initial-3 24 hr-1 24 hr-2 24 hr-3 RH-1RH-2 RH-3 RH-1 RH-2 RH-3 Initial hours months months room temp 6 99.48%99.46% 99.37% 99.36% 99.35% 99.44% 99.29% 99.81% 98.75% 98.88% 98.86%98.89% feasible feasible feasible feasible Remains in (25 C.) (25 C.)(25 C.) Solution 15 96.87% 97.19% 96.88% 96.80% 96.39% 96.84% 96.86%96.57% 96.70% 96.20% 96.18% 96.21% feasible feasible feasible feasibleRemains in Solution 17 98.49% 98.70% 98.78% 98.45% 98.65% 98.82% 98.52%98.29% 98.92% 98.74% 98.75% 98.70% feasible feasible feasible feasibleRemains in (25 C.) (25 C.) (25 C.) Solution 18 99.32% 99.09% 98.97%99.00% 99.14% 99.07% 98.38% 98.39% 98.39% 98.94% 98.54% 98.71% feasiblefeasible feasible feasible Remains in Solution 9 99.38% 99.36% 99.49%99.33% 99.56% 99.44% 99.44% 99.17% 99.49% 99.56% 99.53% 99.23% feasiblefeasible feasible feasible Remains in Solution 59 99.00% 98.88% 98.94%99.03% 98.87% 98.91% feasible feasible Remains in Solution 24 99.63%99.62% 99.64% feasible Remains in Solution 45 99.73% 99.71% 99.68%Feasible Remains in 99.48% 99.38% 99.58% N/A Solution 99.86% 99.63%99.68% N/A 99.73% 99.57% 99.78% N/A 34 95.93% 95.84% 95.91% feasiblePurity of compound in Drug product was 95.9%. Was found to be 99% aftersynthesis. Precipitation at 55 days 7 99.34% 99.31% 99.23% feasibleRemains in Solution 43 98.60% 98.61% 98.49% feasible Remains in Solution9 98.26% 98.14% 97.98% feasible Hazy homogenous appearance, possiblesolution ~24 hr Visible sold precipitant ~24 hr Visible sold precipitant~24 hr Clearly precipitated out of solution ~24 hr 1 100.00% 100.00%100.00% feasible Remains in Solution 3 N/A N/A N/A 99.5% 99.54% 99.34%feasible Remains in (6 (6 (6 Solution days) days) days) 4 99.65% 99.66%99.76% feasible Remains in Solution 5 99.72% 99.71% 99.72% feasibleRemains in Solution 35 100.00% 100.00% 100.00% feasible Remains inSolution

IV. Pharmacokinetic Evaluation

Example 1 Rat Pharmacokinetic Studies

Purpose

The purpose of this study is to determine the pharmacokinetics of testcompounds in plasma, following intramuscular administration to maleSprague Dawley Rats (n=3, unless otherwise specified).

Acclimation/Quarantine

Animals are assessed as to their general health and acclimated for atleast 3 days before being placed on study.

Animal Husbandry

Animals are housed during acclimation and individually housed during thestudy. The animal room environment was controlled (target conditions:temperature 18 to 26° C., relative humidity 30 to 70%, 12 hoursartificial light and 12 hours dark). Temperature and relative humiditywere monitored daily. Water was provided to the animals ad libitum.

Animal Body Weights and Clinical Observation

Body weights were determined before selection to the study and on theday of dose administration. Weight monitoring was done every week.

Detailed clinical observation including behavior and activity,reflection, respiration, skin and fur, facial feature, genitourinarysystem, and other gross lesions was performed on the dosing day and ateach sample collection time point.

Dose Administration

The dose formulation of 400 mg base equivalents/ml in sesame oil+1%benzyl alcohol (unless otherwise specified) was administered byintramuscular injection. The dose volume was determined by the animals'body weight determined on the morning of dosing day.

Sample Collection

Each blood collection (about 0.2 mL per time point) was performed fromjugular vein puncture of each animal into pre-chilled plasticmicrocentrifuge tubes containing 5 μL of 160 mg/mL sodiumfluoride/potassium oxalate (NaF/KO=1/3) with 5% PMSF(100 mM in ethanol)as stabilizer and 4 μL: of EDTA-K2 as anti-coagulant and placed on wetice until centrifugation.

Plasma Processing

Each collected blood sample was centrifuged for 4 minutes at 4° C. and10000 rpm for plasma collection. Plasma was collected and transferredinto a pre-labeled PP tube in dry ice at each time point andprecipitated immediately using ACN at a ratio of 1:4 (plasma:ACN).Centrifuged again (10 minutes, 12000 rpm) and obtain the supernatant.After terminal collection, all supernatant was stored at approximately−80° C. until bioanalysis.

Bioanalytical Method and Sample Analysis

LC-MS/MS methods for the quantitative determination of test compound inbiological matrix were developed. A calibration curve with 8 non-zerocalibration standards were applied for the method including LLOQ (0.05ng/ml). The sample analysis was performed concurrently with a set ofcalibration standards and two sets of QC samples using the LC-MS/MSmethod.

Data Analysis

Plasma concentration versus time data was analyzed by non-compartmentalapproaches using the Phoenix WinNonlin 6.3 software program. C_(max),T_(max), T_(1/2), AUC_((0-t)), AUC_((0-inf)), MRT_((0-t)), MRT_((0-inf))and graphs of plasma concentration versus time profile were prepared.

The dose for nalmefene dodecanoate was determined by allometric scalingto rat from dog doses as previously reported (Gaekens et al, Journal ofControlled Release 232 (2016) 196-202). Terminal half life wasdetermined for active metabolite of select compounds, and is used forestimating duration above minimally effective plasma concentration forthe active metabolite.

TABLE 27 Terminal Nominal Half-life t_(1/2) Half-life t_(1/2)AUC_(0-inf) AUC_(0-inf) Dose (hour) (hour) (ng · h/mL) (ng · h/mL)Compound (mg/kg) Vehicle prodrug nalmefene prodrug nalmefene Nalmefene0.80 saline — 0.87 — 66.0 HCl-IR 59 17 Sesame oil + 1% 15.2 248 10.41026 Benzyl Alcohol 6 80 Sesame oil + 1% 340 569 78.9 3576 BenzylAlcohol 6 123 Sesame oil + 1% 213 425 620 14704 Benzyl Alcohol 6 165Sesame oil + 1% 204 491 637 18876 Benzyl Alcohol 15 80 Sesame oil + 1%1553 639 679 3046 Benzyl Alcohol 15 123 Sesame oil + 1% 884 993 25747232 Benzyl Alcohol 15 165 Sesame oil + 1% 500 451 2789 14019 BenzylAlcohol 17 200 Sesame oil + 1% Prodrug not 266 Prodrug not 16178 BenzylAlcohol detected detected 18 80 Sesame oil + 1% Prodrug not 599 Prodrugnot 8217 Benzyl Alcohol detected detected 18 123 Sesame oil + 1% Prodrugnot 3409 Prodrug not 13658 Benzyl Alcohol detected detected 18 200Sesame oil + 1% Prodrug not 847 Prodrug not 15104 Benzyl Alcoholdetected detected 23 200 Sesame oil + 1% Prodrug not Not assessedProdrug not 27105 (naltrexone Benzyl Alcohol detected detected(naltrexone metabolite) metabolite) 24 80 Cottonseed + 1% 398 1093 1475584 Benzyl Alcohol 24 123 Cottonseed 717 445 547 11283 oil + 1% BenzylAlcohol 24 165 Cottonseed 851 458 453 19031 oil + 1% Benzyl Alcohol 29165 Sesame oil + 1% 175 911 74.8 17313 (naltrexone Benzyl Alcoholmetabolite)

No adverse affect on body weight or clinical observations were noted inany rats across all studies.

Time vs nalmefene concentration data for nalmefene HCL in 1 mg/ml at0.80 mg/kg is provided in Table 28a.

TABLE 28a Mean conc Time (h) Nalmefene (ng/mL) 1.00 29.4 2.00 2.09 4.000.212 8.00 0.0178 12 ND* 24.0 ND *Not detected

Time vs nalmefene concentration data for compound 59 (nalmefenedodecanoate) in 86 mg/ml concentration at 17 mg/kg is provided in Table28b.

TABLE 28b Mean conc Time (h) Nalmefene (ng/mL) 1.00 7.85 2.00 8.10 4.007.45 8.00 6.85 24.0 6.96 48.0 7.92 72.0 5.77 144 1.59 312 0.403 4800.235 648 0.145

Time vs nalmefene concentration data for compound 6 at 80 mg/kg isprovided in Table 29.

TABLE 29 Mean conc Time (h) Nalmefene (ng/mL) 1.00 9.51 2.00 7.11 4.005.48 8.00 5.09 24.0 5.74 48.0 8.48 72.0 7.18 144 3.73 312 3.08 480 2.49648 2.09 816 1.80 984 1.39 1152 1.14 1320 0.833 1488 0.413

Time vs nalmefene concentration data for compound 6 at 123 mg/kg isprovided in Table 30.

TABLE 30 Mean conc Time (h) Nalmefene (ng/mL) 0.250 43.8 1.00 36.3 2.0024.0 4.00 16.7 8.00 12.1 24.0 11.0 48.0 14.2 72.0 10.7 144 9.17 312 13.6480 10.6 648 11.1 816 8.8 984 6.5 1152 4.0 1320 3.2 1488 1.6 1656 1.71824 1.2 1992 0.9 2160 0.8 2328 0.8 2496 0.6 2664 0.5

Time vs nalmefene concentration data for compound 6 at 165 mg/kg isprovided in Table 31.

TABLE 31 Mean conc Time (h) Nalmefene (ng/mL) 0.250 50.4 1.00 48.0 2.0029.2 4.00 19.3 8.00 13.9 24.0 12.3 48.0 14.4 72.0 12.4 144 10.7 312 14.5480 18.1 648 17.1 816 14.2 984 10.5 1152 6.2 1320 4.9 1488 2.8 1656 2.31824 1.8 1992 1.3 2160 1.3 2328 1.1 2496 0.9 2664 0.7

Time vs nalmefene concentration data for compound 15 at 80 mg/kg isprovided in Table 32.

TABLE 32 Mean conc Time (h) Nalmefene (ng/mL) 1.00 7.07 2.00 4.21 4.002.10 8.00 1.42 24.0 1.98 48.0 2.39 72.0 3.25 144 2.32 312 2.18 480 3.03648 2.15 816 0.944 984 0.714 1152 0.745 1320 0.663 1488 0.706 1656 0.6971824 0.514 1992 0.322 2160 0.441

Time vs nalmefene concentration data for compound 15 at 123 mg/kg isprovided in Table 33.

TABLE 33 Mean conc Time (h) Nalmefene (ng/mL) 0.250 18.7 1.00 20.4 2.0013.4 4.00 6.20 8.00 2.74 24.0 1.54 48.0 1.89 72.0 2.00 144 2.97 312 5.64480 7.88 648 6.60 816 5.33 984 3.87 1152 2.41 1320 2.10 1488 2.33 16562.01 1824 1.93 1992 1.50 2160 1.25 2328 1.01 2496 1.18 2664 0.831

Time vs nalmefene concentration data for compound 15 at 165 mg/kg isprovided in Table 34.

TABLE 34 Mean conc Time (h) Nalmefene (ng/mL) 0.250 30.3 1.00 24.1 2.0015.3 4.00 7.60 8.00 2.92 24.0 1.65 48.0 2.43 72.0 2.60 144 3.11 312 6.45480 13.2 648 12.5 816 7.92 984 6.30 1152 5.30 1320 4.54 1488 3.62 16563.24 1824 3.38 1992 2.16 2160 1.62 2328 0.966 2496 0.831 2664 0.903

Time vs nalmefene concentration data for compound 17 at 200 mg/kg isprovided in Table 35.

TABLE 35 Mean conc IM Time (h) Nalmefene (ng/mL) 1.00 249 2.00 299 4.00364 8.00 340 24.0 157 48.0 80.0 72.0 44.3 144 15.5 312 5.09 480 3.04 6483.37 816 1.90 984 1.29 1152 0.737 1320 0.375 1488 0.309

Time vs nalmefene concentration data for compound 18 at 80 mg/kg isprovided in Table 36.

TABLE 36 Mean conc Time (h) Nalmefene (ng/mL) 0.250 5.13 1.00 14.9 2.0022.9 4.00 39.9 8.00 35.6 24.0 36.6 48.0 32.7 72.0 28.2 144 21.5 312 5.60480 2.99 648 1.58 816 1.29 984 1.34 1152 0.99 1320 0.75 1488 0.505 16560.465 1824 0.412 1992 0.383

Time vs nalmefene concentration data for compound 18 at 123 mg/kg isprovided in Table 37.

TABLE 37 Mean conc Time (h) Nalmefene (ng/mL) 0.250 3.98 1.00 15.6 2.0024.4 4.00 43.5 8.00 43.7 24.0 39.2 48.0 29.8 72.0 26.5 144 15.9 312 4.21480 5.59 648 4.73 816 4.01 984 4.26 1152 3.14 1320 3.10 1488 2.44 16562.55 1824 2.55 1992 1.38 2160 2.52 2328 3.11 2496 1.92 2664 2.27

Time vs nalmefene concentration data for compound 18 at 200 mg/kg isprovided in Table 38.

TABLE 38 Mean conc Time (h) Nalmefene (ng/mL) 1.00 32.3 2.00 59.0 4.0064.8 8.00 76.8 24.0 54.2 48.0 46.7 72.0 38.4 144 29.0 312 13.4 480 5.17648 5.51 816 3.54 984 2.90 1152 2.80 1320 2.05 1488 1.77 1656 1.40 18241.68 1992 1.18 2160 1.42 2328 1.43 2496 0.781 2664 1.18 2832 2.34

Time vs naltrexone concentration data for compound 23 at 200 mg/kg isprovided in Table 39.

TABLE 39 Mean conc Time (h) Naltrexone (ng/mL) 1.00 32.3 2.00 655 4.00727 8.00 603 24.0 384 48.0 181 72.0 109

Time vs nalmefene concentration data for compound 24 at 80 mg/kg isprovided in Table 40.

TABLE 40 Mean conc Time (h) Nalmefene (ng/mL) 0.250 1.93 1.00 2.94 2.004.06 4.00 4.69 8.00 4.86 24.0 5.47 48.0 5.43 72.0 5.46 144 3.87 312 3.87480 4.60 648 5.29 816 3.16 984 2.32 1152 2.14 1320 1.45 1488 1.25 16561.05 1824 1.14 1992 1.09 2160 0.971 2328 0.798 2496 0.788 2664 0.719

Time vs nalmefene concentration data for compound 24 at 123 mg/kg isprovided in Table 41.

TABLE 41 Time Mean conc Nalmefene (h) (ng/mL) 0.250 1.89 1.00 2.38 2.003.28 4.00 4.14 8.00 4.32 24.0 6.20 48.0 5.56 72.0 5.03 144 4.54 312 5.42480 9.14 648 10.6 816 8.71 984 6.46 1152 6.57 1320 3.41 1488 2.64 16561.93 1824 1.29 1992 1.13 2160 0.821 2328 0.763 2496 0.457 2664 0.562

Time vs nalmefene concentration data for compound 24 at 165 mg/kg isprovided in Table 42.

TABLE 42 Time Mean conc Nalmefene (h) (ng/mL) 0.250 3.48 1.00 4.90 2.005.23 4.00 6.41 8.00 7.24 24.0 8.44 48.0 8.24 72.0 7.80 144 9.93 312 12.8480 18.0 648 16.6 816 15.3 984 9.14 1152 5.62 1320 5.44 1488 3.62 16563.90 1824 2.69 1992 1.81 2160 1.28 2328 1.34 2496 0.886 2664 0.591

Time vs naltrexone concentration data for compound 29 at 165 mg/kg (at400 mg/ml in sesame oil) is provided in Table 43a.

TABLE 43a Time Mean conc Naltrexone (h) (ng/mL) 0.250 13.8 1.00 30.42.00 39.6 4.00 53.2 8.00 53.1 24.0 46.2 48.0 34.3 72.0 30.4 144 39.6 31228.4 480 13.1 648 6.00 816 4.56 984 3.88 1152 3.71 1320 2.71 1488 2.701656 2.45 1824 2.25 1992 1.92

Time vs naltrexone concentration data for compound 29 (at 300 mg/ml incottonseed oil, n−2) at 165 mg/kg is provided in Table 43b.

TABLE 43b Time Mean conc Naltrexone (h) (ng/mL) 0.250 29.0 1.00 40.12.00 69.3 4.00 91.4 8.00 90.5 24.0 92.1 48.0 66.9 72.0 54.7

Example 2 Dog Pharmacokinetic Studies

Purpose

The purpose of this study is to determine the pharmacokinetics of testcompounds in plasma, following deep intramuscular administration toBeagle dogs (n=3, unless otherwise specified).

Acclimation/Quarantine

Animals are assessed as to their general health and acclimated for atleast 5 days before being placed on study.

Animal Husbandry

Animals are pair housed during acclimation and individually housedduring the study. The room(s) will be controlled and monitored forrelative humidity (targeted mean range 40% to 70%, and any excursionfrom this range for more than 3 hours will be documented as a deviation)and temperature (targeted mean range 18° to 26° C., and any excursionfrom this range will be documented as a deviation) with 10 to 20 airchanges/hour. The room will be on a 12-hour light/dark cycle except wheninterruptions are necessitated by study activities. Animals will be fedtwice daily. Stock dogs will be fed approximately 220 grams of CertifiedDog Diet daily (Beijing Keao Xieli Feed Co., Ltd. Beijing, P.R. China).These amounts can be adjusted as necessary based on food consumption ofthe group or an individual body weight changes of the group or anindividual and/or changes in the certified diet. Reverse osmosis (RO)water is available to all animals, ad libitum. RO water is analyzedevery three months and every batch of feed is analyzed before using.Enrichment toys are provided.

Animal Body Weights and Clinical Observation

Body weights were determined before selection to the study and on theday of dose administration. Weight monitoring was done every week.

Detailed clinical observation including behavior and activity,reflection, respiration, skin and fur, facial feature, genitourinarysystem, and other gross lesions was performed on the dosing day and ateach sample collection time point.

Dose Administration

The dose formulation (concentration−400 mg base equivalents/ml in sesameoil+1% benzyl alcohol, unless otherwise specified) was administered viadeep intramuscularly (unless otherwise specified). The injection vehiclewas also dosed via deep intramuscular route (unless otherwise specified)on contralateral site of each animal at study initiation. The animalswere sedated with Propofol at 6 mg/kg via IV administration. Followingsedation hair was carefully removed from around the injection site andthe area gently cleaned. Care will be taken to avoid irritating skinduring shaving and cleaning the injection site. Then dogs will be dosedwith deep IM administration. At least 2.5 cm depth from the surface intothe central aspect of the quadriceps or biceps femoris muscle, byangling the needle toward the femur. If the needle hits the femur,simply draws back slightly and then inject. The dose volume will bedetermined by the animals' body weight collected on the morning ofdosing day. For repeated administration, the injection sites may berotated to minimize tissue injury.

Sample Collection

Blood samples were collected from a peripheral vessel from restrained,non-sedated animals per sampling time point.

Approximately 0.8 mL blood will be collected at each time point. Allblood samples will be transferred into pre-chilled plasticmicrocentrifuge tubes containing 20 μL of 160 mg/mL sodiumfluoride/potassium oxalate (NaF/KO=1/3) with 5% PMSF(100 mM in ethanol)as stabilizer and 16 μL of EDTA-K2 (0.5M) as anti-coagulant and placedon wet ice until centrifugation.

Each collected blood will be in the wet-ice before centrifuge.

Plasma Processing

Each collected blood sample was centrifuged for 4 minutes at 4° C. and10000 rpm for plasma collection. Plasma was collected and transferredinto a pre-labeled PP tube in dry ice at each time point andprecipitated immediately using ACN at a ratio of 1:4 (plasma:ACN).Centrifuged again (10 minutes, 12000 rpm) and obtain the supernatant.

After terminal collection, all supernatant was stored at approximately−80° C. F until bioanalysis.

Bioanalytical Method and Sample Analysis

LC-MS/MS methods for the quantitative determination of test compound inbiological matrix were developed. A calibration curve with 8 non-zerocalibration standards were applied for the method including LLOQ (0.05ng/ml). The sample analysis was performed concurrently with a set ofcalibration standards and two sets of QC samples using the LC-MS/MSmethod.

Data Analysis

Plasma concentration versus time data was analyzed by non-compartmentalapproaches using the Phoenix WinNonlin 6.3 software program. C_(max),T_(max), T_(1/2), AUC_((0-t)), AUC_((0-inf)), MRT_((0-t)), MRT_((0-inf))and graphs of plasma concentration versus time profile were prepared.

TABLE 44 AUC_(0-inf) Terminal Half- (ng · h/mL) Nominal Half-lifet_(1/2) life t_(1/2) (hour) AUC_(0-inf) nalmefene IM Dose (hour)nalmefene or (ng · h/mL) or Compound (mg/kg) Vehicle prodrug naltrexoneprodrug naltrexone Status 6 30 Sesame oil + 1% Benzyl 95 Data pending398 6100 Ongoing Alcohol 48 Sesame oil + 1% Benzyl 135 277.9 1025 11464Complete (shallow) Alcohol 48 (deep) Sesame oil + 1% Benzyl 54 552.62186 10619 Complete Alcohol 96 Sesame oil + 1% Benzyl 88 Data pending1290 17090 Ongoing Alcohol 15 30 Sesame oil + 1% Benzyl 134 Data pending521 682 Ongoing Alcohol 48 Sesame oil + 1% Benzyl 518 4244.8  1144 15021Ongoing Alcohol 96 Sesame oil + 1% Benzyl 162 Data pending 1353 3789Ongoing Alcohol 24 48 Cottonseed + 1% Benzyl 102 308.8 526 8500 Complete(shallow) Alcohol 48 (deep) Cottonseed + 1% Benzyl 59.6 404.0 613 7226Complete Alcohol 45 30 Cottonseed + 1% Benzyl 151 Data pending 100 13069Ongoing Alcohol 48 Cottonseed + 1% Benzyl 171 Data pending 267 10973Ongoing Alcohol 96 Cottonseed oil + 1% None Data pending None 10215Ongoing Benzyl Alcohol detected detected 7 48 Sesame oil + 1% Benzyl 201Data pending 610 2145 Ongoing Alcohol 8 48 Sesame oil + 1% Benzyl 128Data pending 264 8980 Ongoing Alcohol 1 48 Sesame oil + 1% Benzyl 261Data pending 1040 3708 Ongoing Alcohol 3 48 Sesame oil + 1% Benzyl 63.1Data pending 360 9084 Complete Alcohol 4 48 Sesame oil + 1% Benzyl 28Data pending 829 6535 Complete Alcohol 5 48 Sesame oil + 1% Benzyl 70.9Data pending 3869 8579 Complete Alcohol 35 48 Sesame oil + 1% Benzyl 70Data pending None None Complete Alcohol detected detected

Time vs nalmefene concentration data for compound 6 at 30 mg/kg isprovided in Table 45.

TABLE 45* Time Mean conc Nalmefene (h) (ng/mL) 0.25 8.07 1 11.50 2 7.754 4.33 8 3.41 24 10.14 48 10.82 72 12.15 168 10.20 336 5.24 504 2.86 6723.73 *study is ongoing

Time vs nalmefene concentration data for compound 6 at 48 mg/kg (shallowIM injection) provided in Table 46.

TABLE 46 Time Mean conc Nalmefene (h) (ng/mL) 0.25 4.75 1 5.03 2 3.98 45.26 8 5.40 34 10.25 48 14.50 72 21.40 196 26.80 336 15.37 504 8.34 6725.15 840 2.18 1008 1.82 1176 1.53 1344 1.07 1512 0.53

Time vs nalmefene concentration data for compound 6 at 48 mg/kg (deep IMinjection; redosed in dogs from Table 46) is provided in Table 47a.

TABLE 47a* Time Mean conc Nalmefene (h) (ng/mL) 0.25 8.99 1 18.97 213.23 4 9.70 8 11.08 24 12.91 48 15.83 72 19.93 168 21.93 336 12.00 5045.59 672 4.31 840 2.24 1008 2.07 1176 1.86 1344 1.26 1512 1.11 2424 0.37

Time vs nalmefene concentration data for compound 6 at 48 mg/kg (deep IMinjection; single dose in naïve dogs n=2) is provided in Table 47b.

TABLE 47b* Time Mean conc Nalmefene (h) (ng/mL) 0.25 17.3 1 17.0 2 13.24 9.99 8 7.73 24 12.3 48 17.6 72 25.0 168 13.7 336 6.87 504 5.26 6722.96 *study is ongoing

*study is ongoing

Time vs nalmefene concentration data for compound 6 at 96 mg/kg isprovided in Table 48.

TABLE 48* Time Mean conc Nalmefene (h) (ng/mL) 0.25 13.59 1 24.17 214.11 4 12.56 8 6.84 24 17.80 48 22.13 72 33.97 168 41.9 336 22.2 50413.3 672 7.18 *study is ongoing

Time vs nalmefene concentration data for compound 15 at 30 mg/kg isprovided in Table 49.

TABLE 49* Time Mean conc Nalmefene (h) (ng/mL) 0.25 5.74 1 10.04 2 5.494 2.84 8 0.67 24 0.41 48 0.56 72 0.78 168 0.578 336 0.437 504 0.385 6720.401 *study is ongoing

Time vs nalmefene concentration data for compound 15 at 48 mg/kg isprovided in Table 50a.

TABLE 50a* Time Mean conc Nalmefene (h) (ng/mL) 0.25 6.74 1 15.67 210.28 4 4.23 8 1.19 24 1.65 48 1.95 72 3.14 168 2.92 336 3.94 504 2.71672 1.96 840 1.90 1008 2.10 1176 1.70 1344 2.40 1512 2.44 2064 1.75 22321.58 2400 1.72 2568 1.46 2736 2.06 2904 2.19 3072 1.67 3240 1.32 34081.32 *study is ongoing

Time vs nalmefene concentration data for compound 15 at 48 mg/kg (repeatof study from Table 50a) is provided in Table 50b.

TABLE 50b* Time Mean conc Nalmefene (h) (ng/mL) 0.25 7.55 1 12.3 2 10.34 4.48 8 1.55 24 0.965 48 1.53 72 1.64 168 2.32 336 2.08 504 1.28 6721.47 *study is ongoing

Time vs nalmefene concentration data for compound 15 at 96 mg/kg isprovided in Table 51.

TABLE 51* Time Mean conc Nalmefene (h) (ng/mL) 0.25 18.63 1 24.13 216.83 4 9.81 8 2.84 24 2.38 48 2.65 72 2.98 168 3.61 336 4.06 504 3.03672 2.32 *study is ongoing

Time vs nalmefene concentration data for compound 18 at 48 mg/kg isprovided in Table 52.

TABLE 52 Time Mean conc Nalmefene (h) (ng/mL) 0.25 3.49 1 5.44 2 5.18 46.62 8 9.77 24 14.40 48 14.24 72 16.13

Time vs nalmefene concentration data for compound 24 at 48 mg/kg(shallow IM injection) is provided in Table 53.

TABLE 53 Time Mean conc Nalmefene (h) (ng/mL) 0.25 1.33 1 1.62 2 2.17 44.70 8 4.71 24 8.34 48 10.93 72 15.83 168 24.07 336 11.29 504 5.13 6722.42 840 1.59 1008 1.26 1176 0.86 1344 0.56 1512 0.50

Time vs nalmefene concentration data for compound 24 at 48 mg/kg (deepIM injection) is provided in Table 54.

TABLE 54 Time Mean conc Nalmefene (h) (ng/mL) 0.25 1.64 1 2.03 2 2.11 42.23 8 3.47 24 5.82 48 11.60 72 15.90 168 14.63 336 9.96 504 5.85 6722.47 840 1.99 1008 1.51 1176 1.07 1344 0.85 1512 0.54

Time vs nalmefene concentration data for compound 45 at 30 m/kg isprovided in Table 55.

TABLE 55* Time Mean conc Nalmefene (h) (ng/mL) 0.25 2.82 1 4.04 2 3.24 42.75 8 2.61 24 3.00 48 5.31 72 4.72 168 6.26 336 4.36 504 3.09 672 3.51*study is ongoing

Time vs nalmefene concentration data for compound 45 at 48 mg/kg isprovided in Table 56.

TABLE 56* Time Mean conc Nalmefene (h) (ng/mL) 0.25 1.22 1 2.23 2 1.77 41.75 8 1.95 24 3.05 48 10.00 72 10.05 168 18.87 336 14.83 504 7.43 6723.24 840 4.06 1008 2.49 1176 2.17 1344 2.16 1512 2.18 1680 2.00 18481.39 2016 1.33 2184 1.06 *study is ongoing

Time vs nalmefene concentration data for compound 45 at 96 mg/kg isprovided in Table 57.

TABLE 57* Time Mean conc Nalmefene (h) (ng/mL) 0.25 2.93 1 7.71 2 6.64 44.86 8 3.66 24 7.14 48 11.30 72 9.32 168 17.8 336 16.5 504 6.81 672 5.30*study is ongoing

Time vs naltrexone concentration data for compound 7 at 24 mg/kg (n=2 isprovided in Table 58.

TABLE 58* Time Mean conc Naltrexone (h) (ng/mL) 0.25 3.61 1 5.25 2 2.524 0.82 8 0.29 24 0.61 48 0.61 72 0.67 168 1.75 336 2.29 504 2.44 6722.19 *study is ongoing

Time vs naltrexone concentration data for compound 7 at 48 mg/kg isprovided in Table 59.

TABLE 59* Time Mean conc Naltrexone (h) (ng/mL) 0.25 18.23 1 18.13 29.67 4 3.35 8 1.33 24 1.43 48 2.30 72 2.41 168 1.85 336 1.09 504 1.35672 1.27 840 1.95 1008 1.54 1176 1.02 1344 1.03 1512 1.06 *study isongoing

Time vs naltrexone concentration data for compound 8 at 48 mg/kg isprovided in Table 60.

TABLE 60* Time Mean conc Naltrexone (h) (ng/mL) 0.25 103.20 1 159.67 252.33 4 9.74 8 5.03 24 3.46 48 4.06 72 4.87 168 7.28 336 3.91 504 9.72672 4.59 840 2.52 1008 2.70 1176 1.71 1344 1.63 1512 1.60 *study isongoing

Time vs nalmefene concentration data for compound I at 48 mg/kg isprovided in Table 61.

TABLE 61* Time Mean conc Nalmefene (h) (ng/mL) 0.25 1.90 1 4.24 2 2.26 40.92 8 0.57 24 0.87 48 1.41 72 1.84 168 2.57 336 1.78 504 1.49 672 1.21840 1.64 1008 1.85 1176 1.58 1344 1.24 1512 1.19 *study is ongoing

Time vs nalmefene concentration data for compound 3 at 48 mg/kg isprovided in Table 62.

TABLE 62 Time Mean conc Nalmefene (h) (ng/mL) 0.25 2.54 1 4.48 2 5.37 49.26 8 13.37 24 25.43 48 37.57 72 62.60 168 27.77 336 4.50 504 0.97 6720.34

Time vs naltrexone concentration data for compound 4 at 48 mg/kg isprovided in Table 63.

TABLE 63 Time Mean conc Naltrexone (h) (ng/mL) 0.25 21.90 1 38.67 241.17 4 74.47 8 78.07 24 53.23 48 52.93 72 70.03 168 3.76 336  ND** 504ND 672 ND **ND = none detected

Time vs naltrexone concentration data for compound 5 at 48 mg/kg (n=2)is provided in Table 64.

TABLE 64 Time Mean conc Nalmefene (h) (ng/mL) 0.25 1.12 1 2.75 2 3.55 45.49 8 6.67 24 17.40 48 26.95 72 30.65 168 27.85 336 11.00 504 2.07 6721.06

Time vs naltrexone concentration data for compound 35 at 48 mg/kg isprovided in Table 65.

TABLE 65 Time Mean conc Nalmefene (h) (ng/mL) 0.25 9.57 1 19.07 2 14.504 18.80 8 25.80 24 103.87 48 174.67 72 237.00

Clinical observations for dogs treated with compound 6 at 30 mg/kg areprovided in Table 66.

D1501 D1502 D1503 time point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA Day 0 No obvious clinical No obvious clinical No obvious clinicalNo obvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 1 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 2 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation Day 3 No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical observation observation observation observationobservation observation Day 4 No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical observation observation observation observation observationobservation Day 5 No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinicalobservation observation observation observation observation observationDay 6 No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 7 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day14 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation Day 21 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 28 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 35 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 39 No obvious clinical Noobvious clinical a few erythema on regression of No obvious clinical Noobvious clinical observation observation the inside of the left erythemaand observation observation foreleg escharosis on the outside of thehindleg/ a few erythema on the inside of the right foreleg Day 41 Noobvious clinical No obvious clinical a few erythema on new slight rashesand No obvious clinical No obvious clinical observation observation theinside of the left escharosis on the observation observation forelegoutside of the right leg/a few erythema on the inside of the rightforeleg Day 42 slight rashes on the slight rashes on the a few erythemaon new slight rashes and No obvious clinical No obvious clinical leftthigh and crus outside of right thigh/ the inside of the left escharosison the observation observation slight rashes on the foreleg outside ofthe right right crus leg/a few erythema on the inside of the rightforeleg Day 43 slight rashes on the slight rashes on the a few erythemaon new slight rashes and No obvious clinical No obvious clinical leftthigh and crus outside of right thigh/ the inside of the left escharosison the observation observation slight rashes on the foreleg outside ofthe right right crus leg/a few erythema on the inside of the rightforeleg Day 46 slight rashes and slight rashes and a few erythema on afew erythema on the No obvious clinical No obvious clinical escharosison the left escharosis on the the inside of the left outside of thethigh observation observation thigh and crus outside of right thigh/foreleg, fresh slight rashes on the erythema on the right crus outsideof the thigh Day 48 slight rashes and slight rashes and slight rashes onthe slight rashes and No obvious clinical No obvious clinical escharosisof slight escharosis on the inside of the left escharosis on theobservation observation rashes on the outside outside of right thigh/foreleg/slight rashes outside of the thigh/ of left leg/slight slightrashes on the on the outside of the slight rashes on the rashes on theinside right crus left leg inside of foreleg and outside of the crus Day49 slight rashes and slight rashes and slight rashes on the slightrashes and No obvious clinical No obvious clinical escharosis of slightescharosis on the inside of the left escharosis on the observationobservation rashes on the outside outside of right thigh/ foreleg/slightrashes outside of the thigh/ of left leg/slight slight rashes on the onthe outside of the slight rashes on the rashes on the inside right crusleft leg inside of foreleg and outside of the crus Day 53 slight rashesand Recovery of rashes on No obvious clinical slight rashes on the Noobvious clinical No obvious clinical escharosis of slight the outside ofthe leg observation outside of the leg/ observation observation rasheson the outside slight rashes on the of left leg/slight inside of forelegrashes on the inside and outside of the crus Day 55 escharosis of slightNo obvious clinical No obvious clinical slight rashes on the No obviousclinical No obvious clinical rashes on the outside observationobservation outside of the leg/ observation observation of leftleg/several several rashes on the rashes on the outside inside offoreleg of the crus Day 56 No obvious clinical No obvious clinical Noobvious clinical escharosis of rashes No obvious clinical No obviousclinical observation observation observation on the outside of theobservation observation leg/no obvious clinical observation on theinside of foreleg Day 60 No obvious clinical No obvious clinical Noobvious clinical escharosis of rashes No obvious clinical No obviousclinical observation observation observation on the outside of theobservation observation leg/no obvious clinical observation on theinside of foreleg

Clinical observations for dogs treated with compound 6 at 48 mg/kg(Shallow IM) are provided in Table 67.

time D1001 D1002 D1003 point R-TA L-vehicle R-TA L-vehicle R-TAL-vehicle  8 hr No obvious clinical NA No obvious clinical NA No obviousclinical NA observation observation observation 72 hr No obviousclinical NA No obvious clinical NA Slight swelling NA observationobservation Day 5 No obvious clinical NA Slight swelling NA Largerswelling NA observation Day 7 No obvious clinical NA Slight swelling NALarger swelling NA observation Day 8 Slight swelling, NA Slightswelling, NA Larger swelling, NA induration induration induration Day 9The same with NA The same with NA The same with NA Day 8 Day 8 Day8 Day12 Not obvious NA Not obvious NA Obvious swelling NA swelling andswelling and and induration, no induration, no induration, noinflammation, inflammation, inflammation, painless with painless withpainless with touching touching touching Day 16 Same with Day 15 Noobvious clinical Same with Day 15 No obvious clinical Same with Day 15No obvious clinical observation observation observation Day 17 Moreindurated than No obvious clinical More indurated than No obviousclinical More indurated No obvious clinical the vehicle site observationthe vehicle site observation than the vehicle observation site Day 18The same with No obvious clinical The same with No obvious clinical Thesame with No obvious clinical Day 17 observation Day 17 observation Day17 observation Day 21 No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinicalobservation observation observation observation observation observationDay 22 No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 30 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 35 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation Day 42 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 49 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 56 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 63 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation

Clinical observations for dogs treated with compound 6 at 48 mg/kg (DeepIM; redosed in dogs from Table 67) are provided in Table 68a.

time D1001 D1002 D1003 point L-TA R-vehicle L-TA R-vehicle L-TAR-vehicle 8 hr No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinicalobservation observation observation observation observation observationDay 1 No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 2 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 3 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation Day 4 No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical observation observation observation observationobservation observation Day 5 No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical observation observation observation observation observationobservation Day 6 No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinicalobservation observation observation observation observation observationDay 7 No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 14 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 21 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation Day 28 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 35 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 42 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 49 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 56 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 63 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical 101 observation observation observation observationobservation observation

Clinical observations for dogs treated with compound 6 at 48 mg/kg (deepIM injection; single dose in nave dogs n=2) are provided in Table 68b.

time D1001 D1002 point L-vehicle R-TA L-vehicle R-TA Day 0 No obvious Noobvious vomit about 20 g chyme clinical clinical at 1 hr post doseobservation observation Day 3 No obvious No obvious No obvious Noobvious clinical clinical clinical clinical observation observationobservation observation Day 7 No obvious No obvious No obvious Noobvious clinical clinical clinical clinical observation observationobservation observation Day 14 No obvious No obvious No obvious Noobvious clinical clinical clinical clinical observation observationobservation observation Day 21 No obvious No obvious No obvious Noobvious clinical clinical clinical clinical observation observationobservation observation Day 28 No obvious No obvious No obvious Noobvious clinical clinical clinical clinical observation observationobservation observation Day 35 No obvious No obvious No obvious Noobvious clinical clinical clinical clinical observation observationobservation observation Day 42 No obvious No obvious No obvious Noobvious clinical clinical clinical clinical observation observationobservation observation

Clinical observations for dogs treated with compound 6 at 96 mg/kg areprovided in Table 69.

time D1501 D1502 D1503 point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA Day 0 No obvious clinical No obvious clinical No obvious clinicalNo obvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 1 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 2 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation Day 3 No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical observation observation observation observationobservation observation Day 4 No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical observation observation observation observation observationobservation Day 5 No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinicalobservation observation observation observation observation observationDay 6 No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 7 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 14 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation Day 21 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 35 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 42 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 49 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 56 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation

Clinical observations for dogs treated with compound 15 at 30 mg/kg areprovided in Table 70.

D1501 D1502 D1503 time point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA Day 0 No obvious clinical No obvious clinical No obvious clinicalNo obvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 1 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 2 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation Day 3 No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical observation observation observation observationobservation observation Day 4 No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical observation observation observation observation observationobservation Day 5 No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinicalobservation observation observation observation observation observationDay 6 No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 7 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 14 No obviousclinical No obvious clinical large area of red large area of red Noobvious clinical No obvious clinical observation observation spots onthe outside spots on the outside observation observation and inside ofthe and inside of the leg/ leg/the red spots of the red spots of outsidewere in outsidewere in decrustation decrustation Day 18 No obviousclinical No obvious clinical large area of red large area of red Noobvious clinical No obvious clinical observation observation spots onthe outside spots on the outside observation observation of the leg/thered of the leg/the red spots of outside spots of outside in were indecrustation decrustation and and escharosis escharosis Day 20 Noobvious clinical No obvious clinical regression of red regression of redNo obvious clinical No obvious clinical observation observation spots onthe outside spots on the outside observation observation of the leg andof the leg escharosis/ decrustation scap 1.5 cm * 1.5 cm on the insideof right leg Day 21 No obvious clinical No obvious clinical decrustationon the escharosis on the No obvious clinical No obvious clinicalobservation observation outside of the left outside of the leg/observation observation leg scap 1.5 cm * 1.5 cm on the inside of rightleg Day 25 No obvious clinical No obvious clinical decrustation on thedecrustation on the No obvious clinical No obvious clinical observationobservation outside of the left outside of the right observationobservation leg leg Day 27 No obvious clinical No obvious clinicalescharosis and erythema, escharosis No obvious clinical No obviousclinical observation observation decrustation on the and decrustation onobservation observation outside of the left the outside of theleg/erythema on the right leg/erythema on inside of the left leg theinside of the right leg Day 28 No obvious clinical No obvious clinicaldecrustation on the decrustation on the No obvious clinical No obviousclinical observation observation outside of the left outside of theright observation observation leg leg Day 32 No obvious clinical Noobvious clinical escharosis on the escharosis on the No obvious clinicalNo obvious clinical observation observation outside of the left outsideof the right observation observation leg/erythema on the leg/erythema onthe inside of the left leg inside of the right leg Day 34 No obviousclinical No obvious clinical escharosis on the escharosis and a few Noobvious clinical No obvious clinical observation observation outside ofthe left erythema on the observation observation leg/erythema on theoutside of the right inside of the left leg leg/erythema on the insideof the right leg Day 35 No obvious clinical No obvious clinicalescharosis on the escharosis and a few No obvious clinical No obviousclinical observation observation outside of the left erythema on theobservation observation leg/erythema on the outside of the right insideof the left leg leg/erythema on the inside of the right leg Day 39 Noobvious clinical No obvious clinical recovery for recovery for Noobvious clinical No obvious clinical observation observationescharosis/several escharosis/several observation observation rashes andslight rashes and slight decrustation on the decrustation on the outsideof the left outside of the right leg/several rashes leg/several rasheson on the inside of the the inside of the left left leg leg Day 41 Noobvious clinical No obvious clinical recovery for recovery for Noobvious clinical No obvious clinical observation observationescharosis/several escharosis/several observation observation rashes andslight rashes and slight decrustation on the decrustation on the outsideof the left outside of the right leg/several rashes leg/several rasheson on the inside of the the inside of the left left leg leg Day 42 Noobvious clinical No obvious clinical recovery for recovery for Noobvious clinical No obvious clinical observation observationescharosis/several escharosis/several observation observation rashes andslight rashes and slight decrustation on the decrustation on the outsideof the left outside of the right leg/several rashes leg/several rasheson on the inside of the the inside of the left left leg leg Day 43 Noobvious clinical No obvious clinical recovery for recovery for Noobvious clinical No obvious clinical observation observationescharosis/several escharosis/several observation observation rashes andslight rashes and slight decrustation on the decrustation on the outsideof the left outside of the right leg/several rashes leg/several rasheson on the inside of the the inside of the left left leg leg Day 46 Noobvious clinical No obvious clinical recovery for recovery for Noobvious clinical No obvious clinical observation observationescharosis/several escharosis/several observation observation rashes andslight rashes and slight decrustation on the decrustation on the outsideof the left outside of the right leg/several rashes leg/several rasheson on the inside of the the inside of the left left leg leg Day 49 Noobvious clinical No obvious clinical recovery for recovery for Noobvious clinical No obvious clinical observation observationescharosis/several escharosis/several observation observation rashes andslight rashes and slight decrustation on the decrustation on the outsideof the left outside of the right leg/several rashes leg/several rasheson on the inside of the the inside of the left left leg leg Day 53 Noobvious clinical No obvious clinical Recovery for rashes Recovery forrashes No obvious clinical No obvious clinical observation observationon the leg on the leg observation observation Day 56 No obvious clinicalNo obvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 59 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation

Clinical observations for dogs treated with compound 15 at 48 mg/kg areprovided in Table 71a.

D1501 D1502 D1503 time point L-TA R-vehicle L-TA R-vehicle L-TAR-vehicle befor day 7 No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinicalobservation observation observation observation observation observationday 7 No obvious clinical No obvious clinical Swelling for the Noobvious clinical No obvious clinical No obvious clinical observationobservation whole leg observation observation observation day 8 Noobvious clinical No obvious clinical Swelling at upper No obviousclinical No obvious clinical No obvious clinical observation observationleg/edema at lower observation observation observation leg day 9 Noobvious clinical No obvious clinical Swelling at upper No obviousclinical No obvious clinical No obvious clinical observation observationleg/edema at lower observation observation observation leg/skin rashesat groin day 10 No obvious clinical No obvious clinical Swelling at theNo obvious clinical No obvious clinical No obvious clinical observationobservation upper leg/skin observation observation observation rashes atgroin day 11 No obvious clinical No obvious clinical Swelling 9*7 cm atNo obvious clinical No obvious clinical No obvious clinical observationobservation the upper leg/skin observation observation observationrashes at groin day 12 No obvious clinical No obvious clinical Swelling9*7 cm at No obvious clinical No obvious clinical No obvious clinicalobservation observation the upper leg/skin observation observationobservation rashes at groin day 13 No obvious clinical No obviousclinical Slight swelling at No obvious clinical No obvious clinical Noobvious clinical observation observation upper leg/skin observationobservation observation rashes at groin day 14 No obvious clinical Noobvious clinical Slight swelling at No obvious clinical No obviousclinical No obvious clinical observation observation upper leg/skinobservation observation observation rashes at groin day 15 No obviousclinical No obvious clinical Slight swelling at No obvious clinical Noobvious clinical No obvious clinical observation observation upperleg/skin observation observation observation rashes at groin day 16 Noobvious clinical No obvious clinical Slight swelling at No obviousclinical No obvious clinical No obvious clinical observation observationupper leg/skin observation observation observation rashes at groin day17 No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation day 21 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation day 28 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation day 35 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation day 42 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation day 49 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation day 56 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation day 63 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation day 86 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation day 93 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation day 100 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation day 107 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 114 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 121 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 128 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 135 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 142 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 149 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation

Clinical observations for dogs treated with compound 15 at 48 mg/kg areprovided in Table 71b.

D1001 D1002 D1003 time point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA Day 0 No obvious clinical No obvious clinical No obvious clinicalNo obvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 3 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 7 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation Day 14 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 21 No obvious clinical severalrashes on the several rashes on the several rashes on the several rashon the several rash on the observation outside of the outside of theoutside of the outside of the outside of the right leg left leg rightleg left leg right leg Day 24 a few rashes of the a few rashes on thelots of rashes on the escharosis of the rashes a few rashes of the a fewrashes and left leg outside of the outside of the part on the outside ofthe left leg decrustation on the right leg left leg right leg outside ofthe right leg Day 26 escharosis of the Fading in rashes and lots ofrashes on the lots of rashes on the several rashes on the several rashesand several rashes part escharosis on the outside of the left outside ofthe right outside of the left slight decrustation on the outside of theoutside of the right leg/escharosis of the leg/escharosis of halfleg/escharosis of on the outside of left leg leg half rashes rashesrashes part the right leg Day 28 escharosis of the Fading in rashes andlots of rashes on the lots of rashes on the several rashes on theseveral rashes and several rashes part escharosis on the outside of theleft outside of the right outside of the left slight decrustation on theoutside of the outside of the right leg/escharosis of halfleg/escharosis of half leg/escharosis of on the outside of left legleg/new several rashes rashes rashes the right leg rashes on the rightcrus Day 31 escharosis of the Fading in rashes and escharosis of theescharosis and rashes several rashes on the several rashes and severalrashes part escharosis on the several rashes part on on the outside ofthe leg outside of the left slight decrustation on the outside of theoutside of the right the outside of the left leg/escharosis of on theoutside of left leg leg/new several leg rashes the right leg rashes onthe right crus Day 33 escharosis of the Fading in several recovery forrashes recovery for rashes several rashes on the several rashes andseveral rashes on the rashes and escharosis on the outside of the on theoutside of the outside of the left slight decrustation left leg on theoutside of the left leg/only a few right leg/only a few leg/escharosisof on the outside of leg right leg/new several rash, escharosis andrash, escharosis and rashes rashes on the right decrustationdecrustation crus Day 35 rashes subside rashes subside on the recoveryfor rashes rashes subside on several rashes and several rashes and onthe leg outside of leg/several on the outside of the the leg escharosison the slight decrustation rashes on the right left leg/only a fewoutside of the on the outside of leg crus rash, escharosis and left legdecrustation Day 38 No obvious clinical No obvious clinical No obviousclinical Fading in rashes on No obvious clinical No obvious clinicalobservation observation observation the leg observation observation Day40 No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 42 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 45 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation

Clinical observations for dogs treated with compound 15 at 96 mg/kg areprovided in Table 72.

D1501 D1502 D1503 time point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA Day 0 No obvious clinical No obvious clinical No obvious clinicalNo obvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 1 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 2 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation Day 3 No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical observation observation observation observationobservation observation Day 4 No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical observation observation observation observation observationobservation Day 5 No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical No obvious clinicalobservation observation observation observation observation observationDay 6 No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 7 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical Swelling 8*4 cm observation observationobservation observation observation Day 14 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 21 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 28 No obvious clinical Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation observation observationobservation observation observation Day 35 No obvious clinical Slightrashes on the No obvious clinical No obvious clinical No obviousclinical No obvious clinical observation outside of the observationobservation observation observation hindlimb Day 38 Slight rashes on theSlight rashes on the Slight rashes on the Slight rashes on the Slightrashes on the Slight rashes on the outside of the outside of the outsideof the upper outside of the upper outside of the outside of the hindlimbhindlimb hindlimb hindlimb hindlimb, skin hindlimb, skin exuviatingexuviating Day 40 Slight rashes on the Slight rashes on the Slightrashes on the Slight rashes on the Slight rashes on the Slight rashes onthe outside of the outside of the upper outside of the upper outside ofthe upper outside of the outside of the hindlimb hindlimb were hindlimbwere hindlimb hindlimb, skin hindlimb, skin disappearing, newdisappearing, new exuviating exuviating rashes appeared on rashesappeared on the inside. the inside. Day 42 New rashes appeared Noobvious clinical Slight rashes on the Slight rashes on the Slight rasheson the Slight rashes on the on the iniside of the observation inside ofthe inside of the hindlimb outside of the outside of the hindlimbhindlimb hindlimb hindlimb Day 45 Slight rashes New rashes appearedSlight rashes on the Slight rashes on the Slight rashes on the Slightrashes on the appeared on the on the iniside of the inside of the insideof the hindlimb outside of the outside of the iniside of the hindlimbhindlimb hindlimb, skin hindlimb, some hindlimb exuviating were scabbingDay 47 Slight rashes on the Slight rashes on the Slight rashes on theSlight rashes on the Slight rashes on the Slight rashes on the inisideof the iniside of the inside of the inside of the hindlimb outside ofthe outside of the hindlimb hindlimb hindlimb hindlimb, skin hindlimb,some exuviating were scabbing Day 49 Slight rashes Slight rashes Slightrashes on the Slight rashes on the Slight rashes on the Slight rashes onthe scabbing on the scabbing on the inside of the inside of the hindlimboutside of the outside of the iniside of the iniside of the hindlimbhindlimb, skin hindlimb, some hindlimb hindlimb exuviating were scabbingDay 52 No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical No obvious clinical observationobservation observation observation observation observation Day 54 Noobvious clinical No obvious clinical No obvious clinical No obviousclinical No obvious clinical No obvious clinical observation observationobservation observation observation observation Day 56 No obviousclinical No obvious clinical No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation observation

Clinical observations for dogs treated with compound 18 at 48 mg/kg areprovided in Table 73.

time D1501 D1502 D1562 point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA 8 hr No obvious No obvious clinical No obvious No obvious clinicalAbout 10 mL loose feces at 8 hr post dose clinical observation clinicalobservation observation observation 24 hr No obvious Induration at theupper Red spots at Swelling 15*12 cm at the No obvious clinicalInduration 15*13 cm for clinical leg 18*10 cm/swelling at the upper legupper leg/swelling 8*11 at observation the whole leg with observationthe inner upper leg inner upper leg with redness redness and 6*9 cm/BT:39.1° C. and warmth/swelling and warmth/Induration at the deep rednessat the inner upper leg 9*11 cm calf/BT: 40.1° C. with redness andwarmth/BT: 39.9° C. 48 hr swelling Induration at the upper Red spots atSwelling 15*12 cm at the No obvious clinical Induration 15*13 cm for 3*6cm leg 18*10 cm/swelling at the upper leg upper leg/swelling 8*11 atobservation the whole leg with the inner upper leg inner upper leg withredness redness and 8*9 cm/Red spots at and warmth/swelling andwarmth/Induration at the the 1 'clock black at the calf/BT: 40.0° C.inner upper leg 9*11 cm position/BT: 39.1° C. with redness andwarmth/BT: 39.9° C. 72 hr swelling Induration at the upper Red spots atSwelling 15*12 cm at the No obvious clinical Induration 15*13 cm for 3*6cm leg 18*10 cm/swelling at the upper leg upper leg/swelling 8*11 atobservation the whole leg with the inner upper leg inner upper leg withredness redness and 8*9 cm/Red spots at the 1 and warmth/swelling andwarmth/Induration at the 'clock black at the inner upper leg 9*11 cmposition/BT: 39.1° C./ calf/BT: 38.7° C./ulcerated with redness andwarmth/ thin/inappetence scab 2*4 at inner upper BT: 39.4° C./thin/leg/thin/inappetence inappetence 96 hr swelling lame/serious indurationslight lame/induration of the whole No obvious clinical Induration forthe whole 3*6 cm of the whole leg/edema swelling/red leg/edema at distallower observation leg with redness and at ankle/Red spots at the spotsat the leg/induration and black at warmth/BT: 39.4° C./ 1 'clock upperleg proximal lower thin/inappetence/weight position/BT: 39.1° C./leg/necrosis(black and muscle decreases from 6.52 kg tothin/inappetence/weight atrophy) 1.5*1.5 cm at upper 6.17 kg/bloodystools decreases from 6.71 kg to leg and 6*2 cm at lower leg 6.0kg/bloody stools with red secreta/BT: 39.0° C./thin/ inappetence/weightdecreases from 7.76 kg to 6.86 kg/ bloody stools 120 hr swellinglame/serious induration slight lame/induration of the whole No obviousclinical Induration for the whole 3*6 cm of the whole leg/edemaswelling/red leg/edema at distal lower observation leg with redness andat ankle/Red spots at the spots at the leg/induration and black atwarmth/BT: 39.5° C./ 1 'clock upper leg proximal lower thin position/BT:39.8° C./ leg/necrosis(black and muscle thin/inappetence atrophy)1.5*1.5 cm at upper leg and 6*2 cm at lower leg with red secreta/BT:39.1° C./thin/ inappetence 144 hr swelling lame/serious indurationslight lame/induration of the whole No obvious clinical Induration forthe whole 3*6 cm of the whole leg/edema swelling/red leg/edema at distallower observation leg with redness and at ankle/Red spots at the spotsat the leg/induration and black at warmth/BT: 39.5° C./ 1 'clock upperleg proximal lower thin/6.15 kg position/induration at theleg/necrosis(black and muscle inner upper leg with atrophy) 1.5*1.5 cmat upper deep color and leg and 6*2 cm at lower leg warmth/BT: 39.3° C./with red thin/inappetence/6.18 kg secreta/BT: 39.2° C./thin/inappetence/6.92 kg 168 hr swelling lame/serious induration slightlame/induration of the whole No obvious clinical Induration for thewhole 3*6 cm of the whole leg/edema swelling/red leg/edema at distallower observation leg with redness and at ankle/Red spots at the spotsat the leg/induration and black at warmth/ulceration at 1 'clock upperleg proximal lower upper leg with position/induration at theleg/necrosis(black and muscle 0.5*0.5 cm/ inner upper leg with atrophy)1.5*1.5 cm at upper BT: 38.7° C./thin/ deep color and leg and 6*2 cm atlower leg 5.93 kg warmth/BT: 39.2° C./ with red thin/inappetence/6.13 kgsecreta/BT: 39.6° C./thin/ inappetence/7.01 kg day 8 swellinglame/serious induration slight lame/induration of the whole No obviousclinical Induration for the whole 3*6 cm/ of the whole leg/edemaswelling/red leg/edema at distal lower observation leg with redness andat ankle/ulteration with spots at the leg/induration and black atwarmth/ulceration at red and thick secreta at upper leg proximal lowerupper leg with 0.5*0.5 cm upper leg/induration at leg/necrosis(black andmuscle and redness and the inner upper leg with atrophy) 3*1.5 cm atupper warmth/Induration at the deep color and leg and 6*4 cm at lowerleg front lower leg and warmth/BT: 39.4° C./ with red secreta andfluctuation with red and thin/inappetence/6.11 kg muscle exposure/ thickliquid at the back BT: 39.5° C./thin/ lower inappetence/6.76 kg legBT:38.7° C./thin/ 5.98 kg day 9 swelling lame/serious induration slightlame/induration of the whole No obvious clinical Induration for thewhole 3*6 cm/ of the whole leg/edema swelling/red leg/edema at distallower observation leg with redness and at ankle/3 parts of spots at theleg/induration and black at warmth/ulceration at ulceration with yellowupper leg proximal lower upper leg with 0.5*0.5 cm and thick secreta atleg/necrosis(black and muscle and redness and upper leg/induration atatrophy) 3*1.5 cm at upper warmth/Induration at the the inner upper legwith leg and 6*4 cm at lower leg front lower leg and deep color and withyellow secreta and fluctuation with red and warmth/BT: 38.7° C./ muscleexposure/ thick liquid at the back thin/inappetence/6.09 kg BT: 38.5°C./thin/ lower inappetence/6.79 kg legBT: 38.8° C./thin/ 6.15 kg day 10swelling lame/serious induration No obvious induration of the whole Noobvious clinical Induration for the whole 3*6 cm/ of the whole leg/edemaclinical leg/edema at distal lower observation leg with redness and atankle/3 parts of observation leg/induration and black atwarmth/ulceration at ulceration with yellow proximal lower upper legwith 0.5*0.5 cm and thick secreta at leg/necrosis(black and muscle andredness and warmth upper leg one part of atrophy) 3*1.5 cm at upper andbecoming ulceration with pink and leg and 6*4 cm at lower legscab/Induration at the thick secreta at inner with yellow secreta andfront lower leg and upper leg/induration at muscle exposure/ fluctuationwith red and the inner upper leg with BT: 38.8° C./thin/ thick liquid atthe back deep color and inappctence/6.68 kg lower warmth/BT: 38.3° C./legBT: 38.7° C./thin/ thin/inappetence/6.05 kg 6.13 kg day 11 No obviouslame/serious induration No obvious induration of the whole No obviousclinical Induration for the whole clinical of the whole leg/edemaclinical leg/edema at distal lower observation leg with redness andobservation at ankle/3 parts of observation leg/induration and black atwarmth/ulceration at ulceration with yellow proximal lower upper legwith 0.5*0.5 cm and thick secreta at leg/necrosis(black and muscle andredness and warmth upper leg one part of atrophy) 3*1.5 cm at upper andbecoming ulceration with pink and leg and 6*4 cm at lower legscab/Induration at the thick secreta at inner with yellow secreta andfront lower leg and upper leg/induration at muscle exposure/ fluctuationwith red and the inner upper leg with BT: 38.9° C./thin/ thick liquid atthe back deep color and inappetence/6.83 kg lower warmth/BT: 38.5° C./legBT: 38.7° C./ thin/inappetence/5.99 kg thin/6.13 kg day 12 No obviouslame/serious induration No obvious induration of the whole No obviousclinical Induration for the whole clinical of the whole leg/edemaclinical leg/edema at distal lower observation leg with redness andobservation at ankle/3 parts of observation leg/induration and black atwarmth/ulceration at ulceration with yellow proximal lower upper legwith 0.5*0.5 cm and thick secreta at leg/necrosis(black and muscle andredness and warmth upper leg one part of atrophy) 3*1.5 cm at upper andbecoming scab/thin ulceration at inner upper leg and 6*4 cm at lower legleg/induration at the with yellow secreta and inner upper leg withmuscle exposure/thin/ deep color and warmth/ inappetence/thin/inappetence day 13 No obvious lame/swelling at the No obviousinduration of the whole No obvious clinical Induration for the wholeclinical inner upper leg with clinical leg/necrosis(black and muscleobservation leg/ulceration at upper observation purple color/edema atobservation atrophy) 1.5*1.5 cm at upper leg with scab/6.45 kg ankle/3parts of leg and 5*1.5 cm at lower leg ulceration with yellow withmuscle exposure/6.92 kg and thick secreta at upper leg one part ofulceration with pink and thick secreta at inner upper leg/6.06 kg day 14No obvious lame/swelling at the No obvious induration of the whole Noobvious clinical Induration for the whole clinical inner upper leg withclinical leg/necrosis(black and muscle observation leg/ulceration atupper observation purple color/edema at observation atrophy) 1.5*1.5 cmat upper leg with scab/6.38 kg ankle//3 parts of leg and 5*1.5 cm atlower leg ulceration with yellow with muscle exposure/6.93 kg and thicksecreta at upper leg one part of ulceration with pink and thick secretaat inner upper leg/6.01 kg day 15 No obvious lame/swelling at the Noobvious induration of the whole No obvious clinical Induration for thewhole clinical inner upper leg with clinical leg/necrosis(black andmuscle observation leg/ulceration at upper observation purplecolor/edema at observation atrophy) 1.5*1.5 cm at upper leg with scabankle/73 parts of leg and 5*1.5 cm at lower leg ulceration with yellowwith muscle exposure and thick secreta at upper leg one part ofulceration with pink and thick secreta at inner upper leg

Clinical observations for dogs treated with compound 24 at 48 mg/kg(Shallow IM) are provided in Table 74.

D1001 D1002 D1003 time point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA 2 hr No obvious clinical Swelling No obvious clinical Swelling Noobvious clinical Swelling observation observation observation 8 hr Noobvious clinical Larger swelling No obvious clinical Larger swelling Noobvious clinical Larger swelling observation observation observation 24hr No obvious clinical Larger swelling No obvious clinical Largerswelling and a scab on No obvious clinical Larger swelling observationobservation the edge of the swelling part observation 48 hr No obviousclinical Larger swelling No obvious clinical Larger swelling and a scabon No obvious clinical Larger swelling observation observation the edgeof the swelling part observation 72 hr No obvious clinical Largerswelling No obvious clinical Larger swelling and a scab on No obviousclinical Larger swelling observation observation the edge of theswelling part observation 96 hr No obvious clinical Larger swelling Noobvious clinical Larger swelling and a scab on No obvious clinicalLarger swelling observation observation the edge of the swelling partobservation 120 hr No obvious clinical Larger swelling with No obviousclinical Larger swelling with No obvious clinical Larger swellingobservation induration observation induration and a scab on theobservation with induration edge of the swelling part 144 hr 0.1*0.2cm/few red Larger swelling with 2 yellow scab with Larger swelling withNo obvious clinical Larger swelling site induration 0.8*0.5 cm of each,one induration and a scab on the observation with induration was yellowwith red edge of the swelling part edge 178 hr 4 yellow scabs withinduration/slight red/ 3 yellow scab with induration/5*8 cm wide/0.5- Noobvious clinical induration/5*5 cm 0.1*0.2 cm 5*8 cm wide/ 0.2*0.2 cm0.8 cm depth; 0.3*0.3 cm/red observation wide/0.5-1 cm deptheach/superficial 0.5-0.8 cm depth each/superficial(one scab scab covertwo red site) 192 hr 4 yellow scabs with induration/slight red/ 2 yellowscabs with 0.3*0. l cm/red scab No obvious clinical induration/5*5 cm0.1*0.2 cm 5*8 cm wide/ 0.2*0.2 each; 2 yellow induration/5*8 cmwide/0.5- observation wide/0.5-1 cm each/superficial. 0.5-0.8 cm depthscabs with 0.3*0.5 cm 0.8 cm depth; 0.3*0.3 cm/red depth each scab; 216hr 4 yellow scabs with induration/slight red/ Yellow 0.2*0.1 cm/red Noobvious clinical Induration/5*8 cm 0.1*0.2 cm 4*8 cm wide/0.5-0.8 cmdepth scab/superficial/slight/ scab/shrinking; observation wide/0.5-1 cmdepth each/superficial/ shrinking Induration/4*8 cm wide/0.5- leasionshrinking 0.8 cm depth; two discolorer shin became yellow scab/1*1 cm,2*2 cm 240 hr 4 yellow scabs with induration/7*7 cm wide Yellow 0.2*0.1cm/red No obvious clinical Induration/5*7 cm 0.1*0.2 cmscab/superficial/slight/ scab/shrinking; observation wideeach/superficial/ shrinking Induration/4*8 cm wide/; two leasionshrinking discolorer shin became yellow scab/1*1 cm, 2*2 cm 264 hr 4yellow scabs with Induration/7*7 cm wide/ Yellow 0.1*0.1 cm/red Noobvious clinical Induration/9*3.5 cm 0.1*0.1 cm/scab/superficial/slight/ scab/shrinking; observation wide/each/superficial/ shrinking Induration/7*7 cm wide/; two shrinkingyellow scab/1*1 cm, 2*2 cm/superficial 288 hr Yellow Induration/7*7 cmwide/ The yellow Induration/7*8 cm/; two no significant Induration/9*5.5scab/superficial/ scab/disappeared, skin yellow scab/1*1 cm, abnormal cmwide/ slight/shrinking to repairing; 2*2 cm/superficial a red spot 312hr Yellow Induration/slight red/ skin repairing/the skinInduration/slight red/10*6 cm No obvious clinical Induration/9*5 cmscab/superficial/ 10*6 cm(longest*shortest, of scab site; wide/0.5-0.8cm depth; 3 observation wide/0.5-1.5 cm depth slight/shirnking; shortboot shape) wide/ New yellow yellow scabs/moderate; 1 red 0.5-0.8 cmdepth; Skin scab/superficial/1.5*2 cm Skin rash/0.2*0.2 cm rashes atgroin and discolored/red/3*2 cm/with oxter/moderate some little redspot; 336 hr No obvious clinical Induration/slight red/ yellowInduration/10*6 cm wide; 5 No obvious clinical Induration/9*5 cmobservation 10*6 cm(longest*shortest, scab/superficial/2*2 cm yellowobservation wide short boot shape) wide/Skin scabs(2*1 cm, 2*2 cm, 1*1cm, rashes at groin 3*2 cm, 2*0.5 cm) on the right and one scab 2*2 onthe left/moderate ulcer; 360 hr No obvious clinical Induration/slightred/ yellow Induration/10*6 cm wide; 5 No obvious clinicalInduration/9*5 cm observation 10*6 cm(longest*shortest,scab/superficial/2*2 cm/ yellow observation wide short boot shape)wide/Skin alopecia scabs(2*1 cm, 2*2 cm, 1*1 cm, rashes at groin andoxter 3*2 cm, 2*0.5 cm) on the right and one scab 2*2 on theleft/alopecia 384 hr No obvious clinical Induration/slight red/ yellowInduration, the edge of the No obvious clinical Induration/9*5 cmobservation 9.5*6 cm(longest*shortest, scab/superficial/2*2 cm/induration became observation wide short boot shape) wide/Skin alopeciasoft/10*7 cm wide; 4 yellow rashes at groin and scabs(1*1 cm, 2*2 cm,3*2 cm, oxter/moderate 2*0.5 cm) on the right and one scab 2*2 on theleft/alopecia 408 hr No obvious clinical Induration/slight red/ yellowInduration, the edge of the No obvious clinical Induration/11*6 cmobservation 10*7 cm(longest*shortest, scab/superficial/2*2 cm/induration became observation wide short boot shape) wide/Skin alopeciasoft/10*7 cm wide; 4 yellow rashes at groin scabs(1*1 cm, 2*2 cm, 3*2cm, 2*0.5 cm) on the right and one scab 2*2 on the left/alopecia 432 hrNo obvious clinical Induration/slight red/ yellow Induration, the edgeof the No obvious clinical Induration/10*5 cm observation 10*6cm(longest*shortest, scab/superficial/2*2 cm/ induration becameobservation wide short boot shape) wide/Skin alopecia soft/10*6 cm wide;3 yellow rashes at groin and oxter scabs(1*1 cm, 3*2 cm, 2*0.5 cm) onthe right and one scab 2*2 on the left/alopecia 456 hr No obviousclinical Induration/slight red/ yellow Induration, the edge of the Noobvious clinical Induration/9*6 cm observation 10*6 cm(longest*shortest,scab/superficial/2*2 cm/ induration became observation wide(oval) shortboot shape) wide/Skin alopecia soft/11*7 cm wide(oval); 3 rashes atgroin and oxter yellow scabs(1*1 cm, 3*2 cm, 2*0.5 cm) on the right andone scab 2*2 on the left/alopecia 480 hr No obvious clinicalInduration/slight red/ yellow Induration, the edge of the No obviousclinical Induration/9*6 cm observation 10*7 cm(longest*shortest,scab/superficial/2*2 cm/ induration became observation wide(oval) shortboot shape) wide/Skin alopecia soft/11*7 cm wide(oval); 3 rashes atgroin yellow scabs(1*1 cm, 3*2 and 2*0.5 cm) on the right and one scab2*2 on the left/two red bumps/alopecia 504 hr No obvious clinicalInduration shrink/ alopecia Induration shrink, the edge of No obviousclinical Induration observation 9*3 cm/some induration near theinduration became observation shrink/7*3 cm the right knee, 0.2~0.5soft/9*6 cm wide; 2 yellow wide deep/Skin rashes at groin scabs(1*1 cmand 1.5*0.8 cm) on the right/two red bumps/alopecia 528 hr rashes at thevehcle Induration shrink/ alopecia Induration shrink, the edge of Noobvious clinical Induration injection area 9*3 cm/sotne induration nearthe induration became observation shrink/7*3 cm the right knee, 0.2~0.5soft/8*6 cm wide; 1 yellow wide deep/Skin rashes at groin andscab(0.8*0.7 cm) on the oxter right/two red bumps/alopecia 552 hr rashesbegan to Induration shrink/ alopecia Induration shrink, the edge of Noobvious clinical Induration scab 8*3 cm/some induration near theinduration became observation shrink/7*3 cm the right knee, 0.2~0.5soft/8*5 cm wide; 1 yellow wide deep/Skin rashes at groin andscab(0.8*0.7 cm) on the right/ oxter/scabs 0.8*0.6 cm two redbumps/alopecia 576 hr rashes began to Induration shrink/ alopeciaInduration shrink, the edge of No obvious clinical Induration scab 7*3cm/some induration near the induration became observation shrink/6*3 cmthe right knee, 0.2~0.5 soft/7*5 cm wide;/ wide deep/Skin rashes atgroin/ two red scabs 0.8*0.6 cm bumps/alopecia 600 hr rashes began toInduration shrink/ alopecia Induration shrink, the edge of No obviousclinical Induration scab 7*3 cm/some induration near the indurationbecame observation shrink/6*3 cm the right knee, 0.2~0.5 soft/7*5 cmwide;/two red wide deep/Skin rashes at groin and bumps/alopeciaoxter/scabs 0.8*0.6 cm 624 hr rashes began to Induration shrink/alopecia Induration shrink, the edge of No obvious clinical Indurationscab 7*3 cm/some induration near the induration became observationshrink/6*3 cm the right knee, 0.2~0.5 soft/7*5 cm wide;/two red widedeep/Skin rashes at groin and bumps/alopecia oxter/scabs 0.8*0.6 cm 648hr rashes began to Induration shrink/ No obvious clinical Indurationshrink, the edge of No obvious clinical Induration shrink scab 7*3cm/Skin rashes at groin observation the induration became observationand oxter soft/7*5 cm wide; 672 hr rashes Induration shrink/ rashesInduration shrink, the edge of No obvious clinical Swelling/6.5*3 cm5.5*3 cm/Skin rashes at groin the induration became observation andoxter soft/7*4 cm wide; 696 hr rashes Induration shrink/ rashes shrinkInduration shrink, the edge of No obvious clinical Swelling/5.5*3 cm 5*3cm/Skin rashes at groin the induration became observation and oxtersoft/6.5*4 cm wide; 720 hr rashes Induration shrink/ No obvious clinicalInduration shrink, the edge of No obvious clinical Swelling/5*3 cm 4.5*3cm/Skin rashes at groin observation the induration became observationsoft/5*4 cm wide; 744 hr rashes Induration shrink/ No obvious clinicalInduration shrink, the edge of No obvious clinical Swelling/5*3 cm/ 4*3cm/Skin rashes at groin observation the induration became observationinduration at the and oxter soft/5*4 cm wide; middle of swelling 768 hrrashes Induration shrink/ No obvious clinical Induration shrink, theedge of No obvious clinical Swelling/5*3 cm/ 4*3 cm/Skin rashes at groinobservation the induration became observation induration at the andoxter soft/5*4 cm wide; some middle of rushes were found at the swellingstomach 792 hr rashes Induration shrink/ No obvious clinical Indurationshrink, the edge of No obvious clinical Swelling/5*3 cm/ 4*3 cm/Skinrashes at groin observation the induration became observation indurationat the and oxter soft/5*4 cm wide; some middle of rushes were found atthe swelling stomach 816 hr rashes Induration shrink/ No obviousclinical Induration shrink, the edge of No obvious clinical Swelling/4*3cm/ 4*3 cm/Skin rashes at groin observation the induration becameobservation induration at the and oxter soft/5*3 cm wide; some middle ofrushes were found at the swelling stomach 840 hr rashes Indurationshrink/ Swelling/red/warmth swelling for whole No obvious clinicalSwelling/4*3 cm/ 4*3 cm/Skin rashes at groin leg/red/warmth/BT: 38.5° C.observation rushes at and oxter/BT: 38.5° C. groin/BT: 38.4° C. 864 hrrashes Induration shrink/ Swelling/red/warmth swelling 5*3 cm at upperleg No obvious clinical Swelling/4*3 cm/ 4*3 cm/Skin rashes at groin andswelling for the whole observation rushes at and oxter/BT: 38.7° C.calf/red/warmth/BT: 38.9° C. groin/BT: 38.8° C. 888 hr rashes Indurationshrink/ Swelling/red/warmth swelling 5*3 cm at upper leg No obviousclinical Swelling/4*3 cm/ 4*3 cm/Skin rashes at groin and swelling forthe whole observation rushes at and oxter/BT: 38.0° C.calf/red/warmth/BT: 38.5° C. groin/BT: 38.6° C. 912 hr rashes Indurationshrink/ Swelling/red/warmth swelling 5*3 cm at upper leg No obviousclinical Swelling/4*3 cm/ 3*2 cm/Skin rashes at groin and swelling forthe whole observation rushes at and oxter/BT: 38.6° C.calf/red/warmth/BT: 38.9° C. groin/BT: 38.6° C. 936 hr rashes Indurationshrink/ red/warmth swelling 3*2 cm at upper leg No obvious clinicalSwelling/3*2 cm/ 3*2 cm/Skin rashes at groin and swelling for the wholeobservation rushes at and oxter/BT: 38.2° C. calf/red/warmth/BT: 38.9°C. groin/BT: 38.6° C. 960 hr rashes Induration shrink/ No obviousclinical swelling 3*2 cm at upper leg/ No obvious clinical Swelling/3*2cm/ 3*2 cm/Skin rashes at groin observation BT: 39.0° C. observationrushes at and oxter/BT: 38.5° C. groin/BT: 38.8° C. 984 hr rashesInduration shrink/ No obvious clinical swelling 3*2 cm at upper leg/ Noobvious clinical Swelling/3*2 cm/ 2*2 cm/Skin rashes at groinobservation BT: 39.2° C. observation rashes at and oxter/BT: 38.3° C.groin/BT: 38.9° C. 1008 hr rashes Induration shrink/ No obvious clinicalswelling 3*2 cm at upper leg/ No obvious clinical Swelling/3*2 cm/ 2*2cm/Skin rashes at groin observation BT: 38.9° C. observation rushes atand oxter/BT: 38.6° C. groin/BT: 38.6° C. 1032 hr rashes Skin rashes atgroin and oxter No obvious clinical swelling 3*2 cm at upper leg Noobvious clinical Swelling/2*2 cm/ observation observation rushes atgroin 1056 hr rashes Skin rashes at groin and No obvious clinicalswelling 3*2 cm at upper leg/ No obvious clinical Swelling/2*2 cm/oxter/BT: 38.3° C. observation BT: 38.9° C. observation rushes atgroin/BT: 38.6° C. 1080 hr rashes Skin rashes at groin and oxter Noobvious clinical swelling 3*2 cm at upper leg/ No obvious clinicalSwelling/2*2 cm/ observation BT: 38.8° C. observation rushes atgroin/BT: 38.9° C. 1104 hr rashes Skin rashes at groin and No obviousclinical swelling 2*2 cm at upper leg/ No obvious clinical swellingoxter/BT: 38.6° C. observation BT: 38.9° C. observation shrinking/rashessubsiding/BT: 39.1° C. 1128 hr rashes subsiding Skin rashes at groin andoxter No obvious clinical swelling 2*2 cm at upper leg/ No obviousclinical swelling observation BT: 38.8° C. observation shrinking/rashessubsiding 1152 hr rashes subsiding Skin rashes at groin and oxter Noobvious clinical swelling 2*2 cm at upper leg No obvious clinicalswelling observation observation shrinking/rashes subsiding 1176 hrrashes subsiding Skin rashes at groin and oxter No obvious clinicalswelling shrinking No obvious clinical swelling observation observationshrinking/rashes subsiding 1200 hr rashes subsiding Skin rashes at groinand No obvious clinical swelling shrinking/BT: 38.8 No obvious clinicalswelling oxter/BT: 38.7° C. observation observation shrinking/rashessubsiding/BT: 38.9° C. 1224 hr rashes subsiding Skin rashes at groin andNo obvious clinical swelling shrinking/BT: 39.0 No obvious clinicalswelling oxter/BT: 38.8 observation observation shrinking/rashessubsiding/BT: 39.2 1248 hr No obvious clinical Skin rashes at groin andNo obvious clinical swelling shrinking/BT38.9 No obvious clinicalswelling observation oxter/BT: 38.9 observation observationshrinking/rashes subsiding/BT: 38.8 1272 hr No obvious clinical Skinrashes at groin and oxter No obvious clinical No obvious clinical Noobvious clinical No obvious clinical observation observation observationobservation observation 1296 hr No obvious clinical Skin rashes at groinand oxter No obvious clinical No obvious clinical No obvious clinical Noobvious clinical observation observation observation observationobservation 1512 hr No obvious clinical Skin rashes at groin and oxterNo obvious clinical No obvious clinical No obvious clinical No obviousclinical observation observation observation observation observation

Clinical observations for dogs treated with compound 24 at 48 mg/kg(Deep IM) are provided in Table 75.

D1501 D1502 D1502 time poiut L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA 8 hr No obvious No obvious No obvious No obvious No obvious Noobvious clinical clinical clinical clinical clinical clinicalobservation observation observation observation observation observation24 hr No obvious No obvious No obvious No obvious No obvious No obviousclinical clinical clinical clinical clinical clinical observationobservation/ observation observation observation observation BT: 38.8 48hr No obvious red spot near No obvious No obvious No obvious No obviousclinical the saphenous clinical clinical clinical clinical observationvein of hind observation observation observation observation limb/BT:38.6 72 hr No obvious red spot near No obvious No obvious No obvious Noobvious clinical the saphenous clinical clinical clinical clinicalobservation vein of hind observation observation observation observationlimb/BT: 38.8 96 hr No obvious red spot near No obvious Swelling at theNo obvious No obvious clinical the saphenous clinical whole leg,clinical clinical observation vein of hind observation indurationobservation observation limb/BT: 39.2 11*11 cm at the groin/red/BT: 38.3120 hr No obvious red spot near No obvious Swelling at the No obvious Noobvious clinical the saphenous clinical whole leg, clinical clinicalobservation vein of hind observation induration observation observationlimb 11*11 cm at the subsiding/ groin/red/BT: 38.7 BT: 39.2 144 hr Redspot at Sweling No obvious Swelling at the No obvious swelling at thegroin 8*5 cm at inner clinical whole leg, clinical inner upper leg upperleg with observation induration observation 8*8 cm/BT: 39.5redness/swelling 11*11 cm at the at calf/red groin/red/BT: 38.3 spotnear the saphenous vein of hind limb subsiding/ BT: 39.6 168 hr Red spotat Sweling No obvious Swelling at the Red spot at swelling at the groin8*5 cm at inner clinical whole leg, the groin inner upper leg upper legwith observation induration 8*8 cm/BT: 39.3 redness/swelling 11*11 cm atthe at calf/red groin/red/BT: 38.4 spot near the saphenous vein of hindlimb subsiding/ BT: 39.6 192 hr Red spot at Sweling No obvious Swellingat the Red spot at swelling at the groin 8*5 cm at inner clinical wholeleg, the groin inner upper leg upper leg with observation induration 9*9cm 8*8 cm/BT: 38.0 redness/swelling at the at groin/red/BT: 38.5calf/BT: 39.0 216 hr Red spot at swelling No obvious Swelling at the Redspot at swelling at the groin 8*5 cm/ clinical whole leg, the groininner upper leg BT: 39.0 observation induration 9*9 cm 8*8 cm/BT: 38.8at the groin/red/BT: 38.4 240 hr Red spot at swelling No obviousSwelling 9*9 cm Red spot at swelling at the groin 8*5 cm/ clinical atthe back and the groin inner upper leg BT: 39.0 observation inner upper8*8 cm/BT: 39.2 leg/red/BT: 38.9 264 hr Red spot at swelling No obviousSwelling 9*9 cm Red spot at swelling at the groin 6*5 cm/ clinical atthe back and the groin inner upper leg BT: 38.9 observation inner upper8*8 cm/BT: 38.9 leg/red/BT: 38.3 288 hr Red spot at swelling No obviousSwelling 9*9 cm Red spot at swelling at the groin 6*5 cm/ clinical atthe back and the groin inner upper leg BT: 38.9 observation inner uppersubsiding 8*8 cm/BT: 38.8 leg/red/BT: 38.5 day 13 Red spot at swellingNo obvious Swelling 9*9 cm No obvious swelling at the groin 6*5 cmclinical at the back and clinical inner upper leg observation innerupper leg observation 8*8 cm day 14 Red spot at swelling No obviousSwelling 9*8 cm No obvious swelling at the groin 3*4 cm clinical at theback and clinical inner upper leg observation inner upper legobservation 8*7 cm day 15 Red spot at swelling No obvious Swelling 8*8cm No obvious swelling at the groin 3*4 cm clinical at the back andclinical inner upper leg observation inner upper leg observation 8*7 cmday 16 Red spot at No obvious No obvious Swelling 6*6 cm No obviousswelling at the groin clinical clinical at the back and clinical innerupper leg observation observation inner upper leg observation 6*6 cm day17 Red spot at No obvious No obvious Swelling 6*5 cm No obvious swellingat the groin clinical clinical at the back and clinical inner upper legobservation observation inner upper leg observation 4*5 cm day 18 Redspot at No obvious No obvious Swelling 6*5 cm No obvious swelling at thegroin clinical clinical at the back and clinical inner upper legobservation observation inner upper leg observation 4*5 cm day 19 Redspot at No obvious No obvious Swelling 5*5 cm No obvious swelling at thegroin clinical clinical at the back and clinical inner upper legobservation observation inner upper leg observation 4*5 cm day 20 Redspot at No obvious No obvious Swelling 5*5 cm No obvious swelling at thegroin clinical clinical at the back and clinical inner upper legobservation observation inner upper leg observation 4*5 cm day 21 Redspot at No obvious No obvious Swelling 4*4 cm No obvious swelling at thegroin clinical clinical at the back and clinical inner upper legobservation observation inner upper leg observation 3*3 cm day 22 Redspot at No obvious No obvious Swelling 4*4 cm No obvious swelling at thegroin clinical clinical at the back and clinical inner upper legobservation observation inner upper leg observation 2*2 cm day 23 Redspot at No obvious No obvious Swelling 4*4 cm No obvious No obvious thegroin clinical clinical at the back and clinical clinical observationobservation inner upper leg observation observation day 24 Red spot atNo obvious No obvious Swelling 4*3 cm No obvious No obvious the groinclinical clinical at the back and clinical clinical observationobservation inner upper leg observation observation day 25 Red spot atNo obvious No obvious Swelling 4*3 cm No obvious No obvious the groinclinical clinical at the back and clinical clinical observationobservation inner upper leg observation observation day 28 Red spot atNo obvious No obvious Swelling 3*3 cm No obvious No obvious the groinclinical clinical at the back and clinical clinical observationobservation inner upper leg observation observation day 35 No obvious Noobvious No obvious No obvious No obvious No obvious clinical clinicalclinical clinical clinical clinical observation observation observationobservation observation observation day 42 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation day 49 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation day 56 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation day 63 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation

Clinical observations for dogs treated with compound 45 at 30 mg/kg areprovided in Table 76.

time D1501 D1502 D1503 point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA Day 0 No obvious No obvious No obvious No obvious No obvious Noobvious clinical clinical clinical clinical clinical clinicalobservation observation observation observation observation observationDay 1 No obvious No obvious No obvious No obvious No obvious No obviousclinical clinical clinical clinical clinical clinical observationobservation observation observation observation observation Day 2 Noobvious No obvious No obvious No obvious No obvious No obvious clinicalclinical clinical clinical clinical clinical observation observationobservation observation observation observation Day 3 No obvious Noobvious No obvious No obvious No obvious No obvious clinical clinicalclinical clinical clinical clinical observation observation observationobservation observation observation Day 4 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 5 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 6 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 7 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 14 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 21 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 28 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 35 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 42 No obvious No obvious Noobvious No obvious several rashes several rashes clinical clinicalclinical clinical on the on the outside observation observationobservation observation outside and and inside of inside of left rightthigh leg/several rashes on the left crus Day 43 No obvious No obviousNo obvious No obvious several rashes several rashes clinical clinicalclinical clinical on the on the outside observation observationobservation observation outside and and inside of inside of left rightthigh leg/several rashes on the left crus Day 46 No obvious No obviousNo obvious No obvious several rashes several rashes clinical clinicalclinical clinical on the on the outside observation observationobservation observation outside and and inside of inside of left rightthigh/fresh leg/several rashes on the rashes on the upper thigh leftcrus/fresh rashes on the upper thigh Day 48 No obvious No obvious Noobvious No obvious several rashes several rashes clinical clinicalclinical clinical on the on the outside of observation observationobservation observation outside of right left thigh/severalleg/escharosis rashes on the of several thighs/escharosis rashes on theof several left rashes on the crus/several inside of the leg rashes onthe thighs Day 49 several No obvious No obvious No obvious severalrashes several rashes rashes on clinical clinical clinical on the on theoutside the outside observation observation observation outside and andinside of and inside inside of left right thigh of left leg/severalleg/several rashes on the rashes on thighs the thighs Day 53 No obviousNo obvious No obvious No obvious recovery for recovery for clinicalclinical clinical clinical rashes on the rashes on the leg observationobservation observation observation leg Day 55 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 56 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation

Clinical observations for dogs treated with compound 45 at 48 mg/kg areprovided in Table 77.

time D1501 D1502 D1503 point L-TA R-vehicle L-TA R-vehicle L-TAR-vehicle Day 0 No obvious No obvious No obvious No obvious No obviousNo obvious clinical clinical clinical clinical clinical clinicalobservation observation observation observation observation observationDay 1 No obvious No obvious No obvious No obvious No obvious No obviousclinical clinical clinical clinical clinical clinical observationobservation observation observation observation observation Day 2 Noobvious No obvious No obvious No obvious No obvious No obvious clinicalclinical clinical clinical clinical clinical observation observationobservation observation observation observation Day 3 No obvious Noobvious No obvious No obvious No obvious No obvious clinical clinicalclinical clinical clinical clinical observation observation observationobservation observation observation Day 7 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 14 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 21 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 28 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 35 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 42 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 49 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 56 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 63 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 70 No obvious No obvious Noobvious No obvious Slight rashes on Slight rashes clinical clinicalclinical clinical the outside of on the outside observation observationobservation observation the hindlimb of the hindlimb. Day 73 No obviousNo obvious No obvious No obvious Recovery for Recovery for clinicalclinical clinical clinical rashes, skin rashes, skin observationobservation observation observation exuviating exuviating Day 75 Slightrashes Slight No obvious No obvious Recovery for Recovery for appearedon rashes clinical clinical rashes, slight rashes, slight the outside ofappeared observation observation skin exuviating skin hindlimb. on theexuviating outside of hindlimb. Day 77 Slight rashes Slight New rashesNew rashes New rashes New rashes appeared on rashes appeared on appearedon appeared on appeared on the outside of appeared the inside of theinside of both outside both outside hindlimb. on the the the hindlimb.and inside of and inside of outside of hindlimb. the hindlimb. thehindlimb. hindlimb. Day 80 Slight rashes Slight Slight rashes Slightrashes Recovery for Slight rashes appeared on rashes on the inside onthe inside the rashes on on the inside the outside of appeared of the ofthe the outside and of the hindlimb. on the hindlimb, and hindlimb, andinside of the hindlimb, and outside of new rashes new rashes hindlimb.recovery for hindlimb. appeared on appeared on the rashes on theoutside. the outside. the inside Day 82 a few rash on No obvious a fewrash a few rash on rashes recovery for the outside of clinical on thethe outside of disappeared on the rashes on left leg observation outsideof leg the inside and the outside leg/ leg outside of leg several rasheson the inside of leg Day 84 rashes No obvious escharosis of rashesrashes recovery for disappeared on clinical rashes on the disappeareddisappeared on the rashes on the outside of observation outside of onthe outside the inside and the outside leg/ left leg leg of leg outsideof leg several rashes on the inside of leg Day 87 No obvious No obviousNo obvious No obvious No obvious slight rashes clinical clinicalclinical clinical clinical on the inside observation observationobservation observation observation of leg Day 89 No obvious No obviousNo obvious No obvious No obvious slight rashes clinical clinicalclinical clinical clinical on the inside observation observationobservation observation observation of leg Day 91 No obvious No obviousNo obvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation

Clinical observations for dogs treated with compound 45 at 96 mg/kg areprovided in Table 78.

time D1501 D1502 D1503 point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA Day 0 No obvious No obvious No obvious No obvious No obvious Noobvious clinical clinical clinical clinical clinical clinicalobservation observation observation observation observation observationDay 1 No obvious No obvious No obvious No obvious Liquid feces about 20mL clinical clinical clinical clinical observation observationobservation observation Day 2 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation Day 3 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation Day 4 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation Day 5 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation Day 6 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation Day 7 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation Day 14 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation Day 21 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation Day 28 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation Day 35 No obvious No obvious Rashes on theRashes and No obvious No obvious clinical clinical outside of theexuviate on clinical clinical observation observation hindlimb theoutside observation observation of the hindlimb Day 38 Slight rashesSlight rashes Much rashes on Much No obvious No obvious on the inside onthe outside the outside of rashes on clinical clinical and outside of ofthe the hindlimb, the outside observation observation the hindlimbhindlimb skin exuviating of the hindlimb, skin exuviating Day 40 Slightrashes Slight rashes Much rashes on Much No obvious No obvious on theinside on the outside the outside of rashes on clinical clinical andoutside of of the the hindlimb, the outside observation observation thehindlimb hindlimb skin exuviating of the hindlimb, skin exuviating Day42 Slight rashes Slight rashes Slight rashes on Much No obvious Noobvious on the inside on the inside the outside of rashes on clinicalclinical and outside of and outside of the hindlimb, the outsideobservation observation the hindlimb the hindlimb skin exuviating of thehindlimb, skin exuviating Day 45 Slight rashes Slight rashes Slightrashes on Much No obvious No obvious on the inside on the inside theoutside of rashes on clinical clinical and outside of and outside of thehindlimb, the outside observation observation the hindlimb the hindlimbskin exuviating of the hindlimb, skin exuviating Day 47 Rashes RashesSlight rashes on Much No obvious No obvious scabbing on scabbing on theoutside of rashes on clinical clinical the outside of the outside of thehindlimb, the outside observation observation the hindlimb/ thehindlimb/ skin exuviating of the Slight rashes Slight rashes hindlimb,on the inside on the inside skin of the of the exuviating hindlimbhindlimb Day 49 rashes rashes Slight rashes on slight No obvious Noobvious disappeared on disappeared on the outside of rashes on clinicalclinical the inside and the inside and the hindlimb, the outsideobservation observation outside of the outside of the skin exuviating ofthe leg leg hindlimb, skin exuviating Day 52 No obvious rashes Slightrashes on slight No obvious No obvious clinical disappeared on theoutside of rashes on clinical clinical observation the inside and thehindlimb, the outside observation observation outside of the skinexuviating of the leg hindlimb, skin exuviating Day 54 No obvious Noobvious rashes rashes No obvious No obvious clinical clinicaldisappeared on disappeared clinical clinical observation observation theoutside of on the observation observation the hindlimb outside of thehindlimb Day 56 No obvious No obvious No obvious rashes No obvious Noobvious clinical clinical clinical disappeared clinical clinicalobservation observation observation on the observation observationoutside of the hindlimb Day 59 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation

Clinical observations for dogs treated with compound 7 at 24 mg/kg areprovided in Table 79.

time D1001 D1002 point L-vehicle R-TA L-vehicle R-TA Day 0 No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalobservation observation observation observation Day 1 No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalobservation observation observation observation Day 2 No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalobservation observation observation observation Day 3 No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalobservation observation observation observation Day 4 No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalobservation observation observation observation Day 5 No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalobservation observation observation observation Day 6 No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalobservation observation observation observation Day 7 No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalobservation observation observation observation Day 14 No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalobservation observation observation observation Day 21 No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalobservation observation observation observation Day 28 Left hind leg Theright hind leg a No obvious No obvious lateral small red few red rash,molting, clinical clinical rash, inside a small inside a small amountobservation observation amount of red rash of red rash Day 30 Left hindleg Right leg lateral No obvious No obvious lateral small red have smallred clinical clinical rash, inside a small rash, peeling observationobservation amount of red rash Day 32 Left hind leg ; Right leg lateralNo obvious No obvious lateral small red small red rash, clinicalclinical rash, inside a lot of inside a lot of red observationobservation red rash rash Day 35 Left hind leg ; Right leg lateral Noobvious No obvious lateral small red small red rash, clinical clinicalrash, inside a lot of inside a lot of red observation observation redrash rash Day 37 a small amount of exuviate on the No obvious No obviousexuviate on the outside of leg/a clinical clinical outside of leg/alagre amount of rashes observation observation lagre amount of rashes onthe inside of leg on the inside of leg Day 39 a small amount of exuviateon the No obvious No obvious exuviate on the outside of leg/a clinicalclinical outside of leg/a lagre amount of rashes observation observationlagre amount of rashes on the inside of leg on the inside of leg Day 42a lagre amount of a large amount of No obvious No obvious rashes on theinside rashes on the inside clinical clinical of leg of leg observationobservation Day 44 a small amount of a small amount of No obvious Noobvious rashes on the inside rashes on the inside clinical clinical ofleg of leg observation observation Day 46 a small amount of a smallamount of No obvious No obvious rashes on the inside rashes on theinside clinical clinical of leg of leg observation observation Day 49 asmall amount of a small amount of No obvious No obvious rashes on theinside rashes on the inside clinical clinical of leg of leg, Red rashobservation observation subsided

Clinical observations for dogs treated with compound 7 at 48 mg/kg areprovided in Table 80.

time D1501 D1502 D1503 point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA  8 hr No obvious No obvious No obvious No obvious No obvious Noobvious clinical clinical clinical clinical clinical clinicalobservation observation observation observation observation observation24 hr No obvious No obvious No obvious No obvious No obvious No obviousclinical clinical clinical clinical clinical clinical observationobservation observation observation observation observation Day 2 Noobvious No obvious No obvious No obvious No obvious No obvious clinicalclinical clinical clinical clinical clinical observation observationobservation observation observation observation Day 3 No obvious Noobvious No obvious No obvious No obvious No obvious clinical clinicalclinical clinical clinical clinical observation observation observationobservation observation observation Day 7 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 14 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 21 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 28 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 35 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 42 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 49 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 56 No obvious No obvious Noobvious No obvious several rashes several clinical clinical clinicalclinical on the left leg rashes on the observation observationobservation observation right leg Day 58 No obvious No obvious Noobvious No obvious several rashes several clinical clinical clinicalclinical on the left leg rashes on the observation observationobservation observation right leg Day 61 No obvious No obvious Noobvious No obvious several rashes several clinical clinical clinicalclinical on the left leg rashes on the observation observationobservation observation right leg Day 63 No obvious No obvious Noobvious No obvious escharosis of several clinical clinical clinicalclinical several rashes rashes on the observation observationobservation observation on the outside outside of of left leg/ right legseveral rashes on the inside of left leg Day 65 No obvious No obvious Noobvious No obvious escharosis of recovery for clinical clinical clinicalclinical rashes on the rashes on the observation observation observationobservation outside of inside of left leg/ right leg rashes subside onthe inside of left leg Day 68 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation Day 70 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation Day 72 No obvious No obvious No obvious Noobvious No obvious No obvious clinical clinical clinical clinicalclinical clinical observation observation observation observationobservation observation

Clinical observations for dogs treated with compound 8 at 48 mg/kg areprovided in Table 81.

D1501 D1502 D1503 L-vehicle L-vehicle L-vehicle (Sesameoil +(Cottonseed + (Sesameoil + time 1% Benzyl 1% Benzyl 1% Benzyl pointAlcohol) R-TA Alcohol) R-TA Alcohol) R-TA  8 hr No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation 24 hr No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 2 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 3 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 7 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 14 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 21 No obvious Swelling Noobvious No obvious No obvious No obvious clinical 6 * 6.5 cm clinicalclinical clinical clinical observation observation observationobservation observation Day 28 No obvious Swelling No obvious No obviousNo obvious No obvious clinical 5 * 3.5 cm clinical clinical clinicalclinical observation observation observation observation observation Day35 No obvious Swelling 5 * 3 cm No obvious No obvious No obvious Noobvious clinical clinical clinical clinical clinical observationobservation observation observation observation Day 37 Several rashesSeveral No obvious No obvious No obvious No obvious on the inside rasheson the clinical clinical clinical clinical and outside of outside ofobservation observation observation observation the left the righthindlimb. hindlimb. Swelling 5 * 3 cm. Day 40 Several rashes Several Noobvious No obvious No obvious No obvious on the inside rashes on theclinical clinical clinical clinical and outside of inside andobservation observation observation observation the left outside ofhindlimb, more the right on the inside. hindlimb, more on the inside.Day 42 Several rashes Several Several rashes on Several No obvious Noobvious on the inside rashes on the the outside of the rashes onclinical clinical and outside of inside and left hindlimb the outsideobservation observation the left outside of of the right hindlimb, morethe right hindlimb on the inside hindlimb, more on the inside Day 44Several rashes Several Several rashes on Several No obvious One rash onon the inside of rashes on the the outside of the rashes on clinical theoutside of the left inside and left hindlimb the outside observation theright hindlimb outside of of the right hindlimb the right hindlimbhindlimb, more on the inside Day 47 Rashes on the Rashes on Rashes onthe Rashes on No obvious Rashes on inside of the left the outsideoutside of the the outside clinical the outside of hindlimb were of thehindlimb were of the observation the hindlimb scabbing hindlimbdisappearing hindlimb were were were disappearing disappearing,disappearing rashes on the inside were scabbing Day 49 Slight rashesSlight rashes Slight rashes Slight rashes No obvious No obvious appearedon the appeared on appeared on the appeared on clinical clinical outsideof the the outside outside of the the outside observation observationhindlimb, rashes of the hindlimb of the on the inside hindlimb, rasheshindlimb were scabbing on the inside were scabbing Day 51 Slight rasheson Slight rashes Slight rashes on Slight rashes No obvious No obviousthe outside of on the the outside of the on the clinical clinical theoutside of hindlimb outside of observation observation hindlimb, somethe the new rashes hindlimb, rashes hindlimb, rashes appeared on the onthe and inside inside were exuviate scabbing appeared on the groin Day54 Slight rashes on Recovery for Recovery for the Slight rashes Noobvious No obvious the outside of the rashes rashes on the clinicalclinical the hindlimb outside of observation observation the hindlimb,rashes and exuviate appeared on the groin, some new rashes appeared onthe inside Day 56 Slight rashes New rashes Slight rashes on Slightrashes No obvious No obvious and skin appeared on the outside of theappeared on clinical clinical exuviating on the outside hindlimb bothinside observation observation the outside of of the and outside thehindlimb. of the hindlimb, some hindlimb, rashes new rashes and skinappeared on the exuviating inside. on the groin. Day 58 Slight rashesSlight rashes Slight rashes on Slight rashes No obvious No obvious andskin on the the outside of the on both clinical clinical exuviating onoutside of hindlimb inside and observation observation the outside ofthe hindlimb outside of the the hindlimb, slight hindlimb, rashes rasheson the and skin inside exuviating on the groin Day 61 Slight rashes onRecovery for Slight rashes on Slight rashes No obvious No obvious theinside of the the rashes on the outside of the on both clinical clinicalhindlimb the outside hindlimb, skin inside and observation observationof the exuviating outside of hindlimb the hindlimb, skin exuviating onthe outside, rashes and skin exuviating on the groin Day 63 Recovery forRecovery for Slight rashes on Slight rashes No obvious No obvious therashes on the rashes on the outside of the on both clinical clinical theoutside of the outside hindlimb, skin inside and observation observationthe hindlimb/ of the exuviating outside of rashes on the hindlimb theinside of the hindlimb, skin hindlimb exuviating on the outside, rashesand skin exuviating on the groin Day 65 Recovery for Recovery for Slightrashes on Slight rashes No obvious No obvious the rashes on the rasheson the outside of the on both clinical clinical the outside of theoutside hindlimb, skin inside and observation observation the hindlimb/of the exuviating outside of escharosis of hindlimb the rashes on thehindlimb, skin inside of the exuviating hindlimb on the outside, rashesand skin exuviating on the groin Day 68 rashes scabbing No obviousSlight rashes on Slight rashes No obvious No obvious on the inside ofclinical the outside of the on both clinical clinical the hindlimbobservation hindlimb, skin inside and observation observation exuviatingoutside of the hindlimb, skin exuviating on the outside, rashes and skinexuviating on the groin Day 70 rashes scabbing No obvious No obviousrashes and No obvious No obvious on the inside of clinical clinical skinclinical clinical the hindlimb observation observation exuviatingobservation observation on the groin Day 72 rashes scabbing No obviousNo obvious rashes and No obvious No obvious on the inside of clinicalclinical skin clinical clinical the hindlimb observation observationexuviating observation observation on the inside of the hindlimb andgroin Day 75 rashes No obvious No obvious slight rashes No obvious Noobvious disappeared on clinical clinical and skin clinical clinical theinside of the observation observation exuviating observation observationhindlimb on the inside of the hindlimb and groin

Clinical observations for dogs treated with compound I at 48 mg/kg areprovided in Table 82.

time D1501 D1502 D1503 point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA  8 hr No obvious No obvious No obvious No obvious No obvious Noobvious clinical clinical clinical clinical clinical clinicalobservation observation observation observation observation observation24 hr No obvious No obvious No obvious No obvious No obvious No obviousclinical clinical clinical clinical clinical clinical observationobservation observation observation observation observation Day 2 Noobvious No obvious No obvious No obvious No obvious No obvious clinicalclinical clinical clinical clinical clinical observation observationobservation observation observation observation Day 3 No obvious Noobvious No obvious No obvious No obvious No obvious clinical clinicalclinical clinical clinical clinical observation observation observationobservation observation observation Day 7 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 14 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 21 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 28 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 35 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 42 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 49 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 56 No obvious No obvious Noobvious No obvious several rashes No obvious clinical clinical clinicalclinical on the left leg clinical observation observation observationobservation observation Day 58 No obvious No obvious No obvious Noobvious several rashes No obvious clinical clinical clinical clinical onthe outside clinical observation observation observation observation ofleft leg observation Day 61 No obvious No obvious No obvious No obviousseveral rashes No obvious clinical clinical clinical clinical andescharosis clinical observation observation observation observation onthe outside observation of left leg Day 63 No obvious No obvious Noobvious No obvious escharosis of No obvious clinical clinical clinicalclinical rashes on the clinical observation observation observationobservation outside of left observation leg Day 65 No obvious No obviousNo obvious No obvious recovery for No obvious clinical clinical clinicalclinical rashes on the clinical observation observation observationobservation outside of left observation leg Day 68 No obvious No obviousNo obvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 70 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation Day 72 No obvious No obvious Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical clinical clinical observation observation observationobservation observation observation

Clinical observations for dogs treated with compound 3 at 48 mg/kg areprovided in Table 83.

D1001 D1002 D1003 time point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA 8 hr No obvious No obvious No obvious Swelling No obvious Swelling4 * 3 clinical clinical clinical 4 * 3.5 clinical observationobservation observation observation Day 1 spotted swelling/4 * 4.5 cm/No obvious Induration/7 * spotted slight Induration/8 * 5.5 cm/ slightred BT: 39.2° C./ clinical 5 cm/obvious red skin obvious bulge/red skin2 * 2 cm observation bulge/BT: 39.1° C. skin/higher red spot attemperature than left the edge of injection shaving area side/BT: 39.6°C. Day 2 swelling/5 * Induration/8 * Swelling/3 * 3 cm Induration/7 *Swelling/3 * 3 cm/ Induration/8 * 5.5 * 1 cm/ 6 cm/spotted 11 * 1 cm/1 *7 * 1.5 cm/obvious spotted obvious bulge/red slight red 1 red spotbulge/BT: 39.3° C. slight red skin skin/higher skin near the temperaturethan left injection injection site/3 * 3 red side/BT: 39.7° C. spot withyellow discharge at edge of shaving area/BT: 38.9° C. Day 3 swelling/5 *Induration/8 * Swelling/3 * 3 cm/ Induration/9 * Swelling/3 * 3 cm/Induration/7 * 11 * 1 cm/ 6 cm/spotted 11 * 1 cm/3 * spotted 11 * 1.5cm/obvious spotted obvious bulge/red slight red 3 red spot slight redskin bulge/BT: 39.3° C. slight red skin skin/higher skin with yellowtemperature than left discharge at injection edge of side/BT: 39.2° C.shaving area/BT: 39.1° C. Day 4 swelling/5 * Induration/10 *Swelling/3 * 4 cm/ Induration/9 * Swelling/3 * 3 cm/ Induration/8 * 12 *1.5 cm/ 6 cm/spotted 11 * 1 cm/3 * spotted 11 * 1.5 cm/obvious spottedobvious slight red 3 red spot slight red skin bulge/BT: 38.5° C. slightred skin bulge/red skin/ skin with yellow higher temperature dischargeat than left injection edge of side/another shaving induration 4 cm toarea/BT: 38.5° C. the injection site/BT: 38.7° C. Day 5 swelling/5 *Induration Swelling/2.5 * Induration/9 * Swelling/2 * 3.5 cm/ Indurationfor the 6 cm/spotted for the whole 3.5 cm/spotted 10 * 2 cm/obviousspotted whole upper slight red upper leg/ slight red skin bulge/slightslight red skin leg/slight swelling skin edema for warmth/BT: 38.7° C.for the whole the whole calf/another calf/ induration and tenderness,obverious bulge fluctuance, 4 cm to the injection warmth/1.5 * site/3 *3 cm red spot 2 red spot behind the upper leg with yellow with slightdischarge at swelling/BT: 39.0° C. edge of shaving area/BT: 38.6° C. Day6 swelling/5 * Induration Swelling/2 * 2 cm/ Induration/9 * Swelling/2 *3.5 cm/ Induration for the 6 cm/spotted for the whole spotted 10 * 2cm/obvious spotted whole upper slight red upper leg slight red skinbulge/slight slight red skin leg/slight swelling skin/ with slightwarmth/BT: 38.8° C. for the whole swelling red/edema calf/another forwhole for the whole induration and calf calf/ obverious bulgetenderness, 4 cm to the injection fluctuance, site/3 * 3 cm red spotwarmth/1.5 * behind the upper leg 2 red spot with slight with yellowswelling/BT: 39.2° C. discharge at edge of shaving area/BT: 38.4° C. Day7 swelling/4 * Induration Swelling/2 * 2 cm/ Induration/9 * Swelling/2 *3.5 cm/ Induration for the 4 cm/spotted for the whole spotted 10 * 2cm/obvious spotted whole upper slight red upper leg slight red skinbulge/slight slight red skin leg/slight swelling skin/ with slightwarmth/BT: 38.6° C. for the whole swelling red/swelling calf/another forwhole for the whole induration and calf calf/red/ obverious bulgewarmth/1 * 1 4 cm to the injection red spot with site/3 * 3 cm red spotyellow behind the upper leg discharge at with slight edge ofswelling/BT: 39.5° C. shaving area/BT: 38.4° C. Day 8 swelling/4 *Induration Swelling/2 * 2 cm/ Induration/9 * Swelling/2 * 3.5 cm/Induration for the 4 cm/spotted for the whole spotted 10 * 2 cm/obviousspotted whole upper slight red upper leg slight red skin bulge/slightslight red skin leg/slight swelling skin/ with slight warmth/BT: 38.7°C. for the whole swelling red/swelling calf/another for whole for thewhole induration and calf calf/red/ obverious bulge warmth/1 * 1 4 cm tothe injection red spot with site/3 * 3 cm red spot yellow behind theupper leg discharge at with slight edge of swelling/BT: 39.4° C. shavingarea/BT: 38.7° C. Day 9 swelling/4 * Induration Swelling Induration/9 *Swelling/2 * 3.5 cm/ Induration/10 * 9 * 1.5 cm 4 cm/spotted 11.5 * 9 cmat shrinking 10 * 2.5 cm/obvious spotted at the injection slight red theupper leg bulge/4 slight red skin site/slight swelling skin/ with slightscabs at 5 for the whole swelling red and o'clock 2 cm calf/another forwhole warmth/slight near the induration and calf swelling for indurationobverious bulge the whole and one fell 4 cm to the injection calf/BT:38.7° C. off/sight site/upper leg with red/swelling slight at the wholeswelling/BT: 38.8° C. calf/BT: 38.3° C. Day 10 swelling/4 * IndurationSwelling Induration/9 * Swelling/2 * 3.5 cm/ Induration/10 * 9 * 1.5 cm4 cm/spotted 11 * 9 cm at shrinking 10 * 2.5 cm/obvious spotted at theinjection slight red the upper leg bulge/4 slight red skin site/slightswelling skin/ with slight scabs at 5 for the whole swelling red ando'clock 2 cm calf/another for whole warmth/slight near the indurationand calf swelling for induration obverious bulge the whole and two scabs4 cm to the injection calf/BT: 38.4° C. fell off/sight site/upper legwith red/swelling slight at the whole swelling/BT: 38.9° C. calf/BT:38.3° C. Day 11 swelling/4 * Induration Swelling Induration/9 * SwellingInduration and 4 cm/spotted 9 * 6 cm at the shrinking 10 * cm/3warmth/10 * 9 * 1.5 cm slight red upper leg yellow scabs at theinjection skin/ with slight at 5 o'clock site/another swelling red and 2cm near the induration and for whole warmth/slight induration obveriousbulge calf swelling for and two scabs 4 cm to the injection the wholefell off/sight site/upper leg with calf/BT: 38.8° C. red/BT: 38.9° C.slight swelling/BT: 39.4° C. Day 12 swelling/4 * Induration SwellingInduration/9 * Swelling Induration and 4 cm 9 * 6 cm at the shrinking 10cm/3 warmth/10 * 9 * 1.5 cm upper leg yellow scabs at the injection withslight at 5 o'clock site/another red and 2 cm near the induration andwarmth/BT: induration obverious bulge 38.0° C. and two scabs 4 cm to theinjection fell off/sight site/3 * 3 cm red spot red/BT: 38.5° C. behindthe upper leg with slight swelling/BT: 39.3° C. Day 13 swelling/4 *Induration Swelling Induration/9 * Swelling Induration and 4 cm 9 * 5 cmat the shrinking 10 cm/slight warmth/10 * 9 cm at upper legred/warmth/BT: the injection with slight 38.8° C. site/another red andinduration and warmth/BT: obverious bulge 38.4° C. 4 cm to the injectionsite/3 * 3 cm red spot behind the upper leg with slight swelling/BT:39.9° C. Day 14 swelling/4 * Induration No obvious Induration/8 * Noobvious Induration and 4 cm 9 * 5 cm at the clinical 8 cm/slightclinical warmth/8 * 9 cm at dosing site observation red/warmth/BT:observation the injection 38.3° C. site/another induration and obveriousbulge 4 cm to the injection site/upper leg with slight swelling/BT:39.6° C. Day 15 swelling/4 * Induration No obvious Induration/8 * Noobvious Induration and 4 cm shrinking clinical 8 cm/slight clinicalwarmth/8 * 9 cm at 9 * 5 cm at the observation red/warmth/BT:observation the injection dosing site/ 37.8° C. site/another BT: 37.8°C. induration and obverious bulge 4 cm to the injection site/upper legwith slight swelling/BT: 39.4° C. Day 16 swelling/4 * Induration Noobvious Induration/8 * No obvious Induration and 4 cm shrinking clinical8 cm/slight clinical warmth/8 * 9 cm at 9 * 5 cm at the observationred/warmth observation the injection dosing site site/slight swellingfor the whole calf/another induration and obverious bulge 4 cm to theinjection site/upper leg with slight swelling/BT: 39.6° C. Day 17swelling/4 * Induration No obvious Induration/8 * No obvious Indurationand 4 cm shrinking clinical 8 cm/BT: 38.7° C. clinical warmth/8 * 9 cmat 9 * 5 cm at the observation observation the injection site/ dosingsite/ upper leg with BT: 38.3° C. slight swelling/BT: 39.5° C. Day 18Swelling Induration No obvious Induration/8 * No obvious Induration andshrinking shrinking clinical 8 cm/BT: 38.8° C. clinical warmth/8 * 9 cmat 9 * 5 cm at the observation observation the injection site/ dosingsite/ upper leg with BT: 38.4° C. slight swelling/BT: 39.5° C. Day 19Swelling Induration No obvious Induration/8 * No obvious Induration andshrinking shrinking clinical 8 cm/BT: 38.7° C. clinical warmth/8 * 9 cmat 9 * 5 cm at the observation observation the injection site/ dosingsite/ upper leg with BT: 38.3° C. slight swelling/BT: 39.4° C. Day 20Swelling Induration No obvious Induration/8 * No obvious Induration andshrinking shrinking clinical 8 cm/BT: 38.6° C. clinical warmth/8 * 9 cmat 9 * 5 cm at the observation observation the injection site/ dosingsite/ upper leg with BT: 37.9° C. slight swelling/BT: 39.6° C. Day 21Swelling Induration No obvious Induration No obvious Induration andshrinking shrinking clinical shrinking/8 * 8 cm/ clinical warmth/8 * 9cm at 9 * 5 cm at the observation warmth/BT: observation the injectionsite/ dosing site/ 38.5° C. upper leg with BT: 38.6° C. slightswelling/BT: 39.3° C. Day 22 Swelling Induration No obvious IndurationNo obvious Induration and shrinking shrinking clinical shrinking/8 * 8cm/ clinical warmth/8 * 9 cm at 8 * 5 cm at the observation warmth/BT:observation the injection site/ dosing site/ 39.3° C. upper leg with BT:38.8° C. slight swelling/BT: 39.9° C. Day 23 Swelling Induration Noobvious Induration No obvious Induration and shrinking shrinkingclinical shrinking/8 * 8 cm/ clinical warmth/8 * 9 cm at 8 * 5 cm at theobservation warmth/BT: observation the injection site/ dosing site/38.8° C. upper leg with BT: 38.3° C. slight swelling/BT: 39.6° C. Day 24No obvious Swelling No obvious Swelling No obvious Swelling and clinical6 * 3 cm at the clinical shrinking/6 * 8 cm/ clinical warmth/6 * 6 cm atobservation dosing site/ observation warmth/BT: observation theinjection site/ BT: 38.5° C. 39.1° C. BT: 39.5° C. Day 25 No obviousSwelling No obvious Swelling No obvious Swelling and clinical 6 * 3 cmat the clinical shrinking/6 * 8 cm/ clinical warmth/6 * 6 cm atobservation dosing site/ observation warmth/BT: observation theinjection site/ BT: 38.4° C. 38.6° C. BT: 39.8° C. Day 26 No obviousSwelling No obvious Swelling No obvious Swelling 6 * 6 cm at clinical5 * 3 cm at the clinical shrinking/6 * 8 cm/ clinical the injectionsite/ observation dosing site/ observation BT: 38.6° C. observation BT:39.3° C. BT: 38.2° C. Day 27 No obvious Swelling No obvious Swelling Noobvious Swelling 5 * 6 cm at clinical 5 * 3 cm at the clinicalshrinking/6 * 6 cm/ clinical the injection site/ observation dosingsite/ observation BT: 38.6° C. observation BT: 38.9° C. BT: 38.4° C. Day28 No obvious No obvious No obvious Swelling No obvious Swelling3 * 5 cmat clinical clinical clinical shrinking/3 * 5 cm clinical the injectionsite observation observation observation observation Day 29 No obviousNo obvious No obvious Swelling No obvious Swelling3 * 3 cm at clinicalclinical clinical shrinking/3 * 5 cm clinical the injection siteobservation observation observation observation Day 30 No obvious Noobvious No obvious Swelling No obvious Swelling3 * 3 cm at clinicalclinical clinical shrinking/3 * 3 cm clinical the injection siteobservation observation observation observation Day 31 No obvious Noobvious No obvious Swelling No obvious No obvious clinical clinicalclinical clinical shrinking/3 * 3 cm clinical observation observationobservation observation observation Day 32 No obvious No obvious Noobvious Swelling No obvious No obvious clinical clinical clinicalclinical shrinking/3 * 3 cm clinical observation observation observationobservation observation

Clinical observations for dogs treated with compound 4 at 48 mg/kg areprovided in Table 84.

time D1501 D1502 D1503 point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA 8 hr No obvious No obvious No obvious No obvious clinical Noobvious No obvious clinical clinical clinical observation clinicalclinical observation observation observation observation observation Day1 Slight Induration No obvious Swelling at the No obvious Induration forthe swelling for the clinical upper leg 6 * 7 cm clinical whole legwhole leg/ observation observation warmth/lame Day 2 Slight IndurationNo obvious Swelling at the No obvious Induration for the swelling forthe clinical upper leg 6 * 7 cm, clinical whole leg/warms whole leg/observation induration at the observation warmth/lame inner upper leg7 * 7 cm/warmth Day 3 Slight Induration No obvious Swelling at the Noobvious Induration for the swelling for the clinical upper leg 6 * 7 cm,clinical whole leg/warms whole leg/ observation induration at theobservation warmth/lame inner upper leg 7 * 7 cm/warmth Day 5 SlightInduration No obvious Swelling at the No obvious Induration for theswelling for the clinical upper leg 6 * 7 cm, clinical whole leg/warmsupper observation induration at the observation leg/warmth inner upperleg 7 * 7 cm/warmth/scap 1 * 0.5 cm at the right of vulva Day 7 SlightInduration No obvious Swelling at the No obvious Induration for theswelling for the clinical upper leg 3 * 4 cm, clinical whole leg/warmsupper observation induration at the observation leg/warmth inner upperleg 7 * 7 cm/warmth/scap 1 * 0.5 cm at the right of vulva Day 10 Noobvious Induration No obvious Induration at the No obvious Indurationfor the clinical for the clinical upper leg/Scap at the clinical wholeleg/warms observation upper leg observation vulva fell off observationDay 12 No obvious Induration No obvious Induration at the No obviousInduration at the clinical for the clinical upper leg clinical upper leg9 * 7 cm observation upper leg observation 7 * 4 cm/Scap at theobservation vulva fell off Day 17 No obvious Induration No obviousInduration at the No obvious Induration at the clinical for the clinicalupper leg 7 * 4 cm clinical upper leg 9 * 6 cm observation upper legobservation observation Day 19 No obvious Induration No obviousInduration at the No obvious Induration at the clinical at the upperclinical upper leg 7 * 4 cm clinical upper leg observation leg 8 * 5 cmobservation observation 7 * 4.5 cm Day 21 No obvious Induration Noobvious Induration at the No obvious No obvious clinical at the upperclinical upper leg 4 * 4 cm clinical clinical observation leg 8 * 4 cmobservation observation observation Day 24 No obvious Induration Noobvious Induration at the No obvious No obvious clinical at the upperclinical upper leg 3.5 * 4 cm clinical clinical observation legobservation observation observation 7.5 * 4 cm Day 26 No obviousInduration No obvious No obvious clinical No obvious No obvious clinicalat the upper clinical observation clinical clinical observation leg 6 *4 cm observation observation observation Day 28 No obvious Induration Noobvious No obvious clinical No obvious No obvious clinical at the upperclinical observation clinical clinical observation leg 6 * 4 cmobservation observation observation Day 31 No obvious Induration Noobvious No obvious clinical No obvious No obvious clinical at the upperclinical observation clinical clinical observation leg 6 * 4 cmobservation observation observation Day 33 No obvious Induration Noobvious No obvious clinical No obvious No obvious clinical at the upperclinical observation clinical clinical observation leg observationobservation observation

Clinical observations for dogs treated with compound 5 at 48 mg/kg areprovided in Table 85.

time D1001 D1002 point L-vehicle R-TA L-vehicle R-TA  72 hr No obviousSwelling Swelling Induration clinical 2*2.5 cm 2*3 cm 6.5 cm*4.5 cmobservation  96 hr No obvious Swelling Swelling Induration clinical 3*3cm 4.5*2.5 cm 6 cm*8 cm with observation slight red color 120 hr Noobvious Swelling Swelling Induration clinical 3*3 cm 4.5*2.5 cm 6 cm*8cm with observation slight red color 144 hr No obvious Swelling SwellingInduration clinical 3.5*3 cm 4.5*2.5 cm 6 cm*8 cm observation 168 hr Noobvious Induration Swelling Induration clinical 4*3 cm 4.5*2.5 cm 6 cm*8cm observation 192 hr No obvious Induration Swelling Induration clinical4*3 cm 4.5*2.5 cm 6 cm*8 cm observation 216 hr No obvious IndurationSwelling Induration clinical 4*3 cm 4.5*2.5 cm 6 cm*8 cm observation 240hr No obvious Swelling Swelling Induration clinical 2*3 cm/a 3.5*2 cm 7cm*8 cm observation swellen with pus at groin 264 hr No obvious Noobvious Swelling Induration clinical clinical 2*2 cm 7 cm*8 cm/warmth/observation observation/ BT: 39.5° C. BT: 38.6° C. 288 hr No obvious Noobvious Swelling Induration clinical clinical 2*2 cm 6 cm*8 cm/observation observation/ BT: 39.5° C. BT: 38.6° C. 312 hr No obvious Noobvious Swelling Induration clinical clinical 2*2 cm shrinking/observation observation/ 6 cm*8 cm/ BT: 38.0° C. BT: 38.5° C. 336 hr Noobvious No obvious Slight Induration clinical clinical swellingshrinking observation observation/ 2*2 cm 3 cm*4 cm/ BT: 38.4° C. BT:38.7° C. 360 hr No obvious No obvious No obvious Induration clinicalclinical clinical shrinking observation observation/ observation 3 cm*4cm/ BT: 38.1° C. BT: 39.1° C. 384 hr No obvious No obvious No obviousInduration clinical clinical clinical shrinking observation observation/observation 3 cm*4 cm/ BT: 38.7° C. BT: 39.3° C. 408 hr No obvious Noobvious No obvious Induration clinical clinical clinical shrinkingobservation observation observation 3 cm*4 cm/ BT: 39.4° C. 432 hr Noobvious No obvious No obvious Swelling clinical clinical clinical 3 cm*3cm/ observation observation/ observation BT: 38.9° C. BT: 38.0° C. 456hr No obvious No obvious No obvious Swelling clinical clinical clinical3 cm*3 cm/ observation observation observation BT: 38.9° C. 480 hr Noobvious No obvious No obvious Swelling clinical clinical clinical 3 cm*3cm/ observation observation/ observation BT: 39.2° C. BT: 38.0° C. 504hr No obvious No obvious No obvious Swelling clinical clinical clinical3 cm*3 cm/ observation observation observation BT: 38.7° C. 528 hr Noobvious No obvious No obvious Swelling clinical clinical clinical 3 cm*3cm/ observation observation/ observation BT: 37.9° C. BT38.2° C. 552 hrNo obvious No obvious No obvious Swelling clinical clinical clinical 3cm*3 cm/ observation observation/ observation BT: 39.2° C. BT: 38.4° C.576 hr No obvious No obvious No obvious Swelling clinical clinicalclinical 3 cm*3 cm/ observation observation/ observation BT: 39.0° C.BT: 38.4° C. 600 hr No obvious No obvious No obvious Swelling clinicalclinical clinical 3 cm*3 cm/ observation observation/ observation BT:38.6° C. BT: 38.3° C. 624 hr No obvious No obvious No obvious Swellingclinical clinical clinical 3 cm*3 cm/ observation observation/observation BT: 38.9° C. BT: 38.6° C. 648 hr No obvious No obvious Noobvious Swelling clinical clinical clinical 3 cm*3 cm/ observationobservation/ observation BT: 38.5° C. BT: 38.3° C. 672 hr No obvious Noobvious No obvious Swelling clinical clinical clinical 3 cm*3 cm/observation observation/ observation BT: 39.3° C. BT: 38.3° C. 696 hr Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical observation observation observation observation 720 hr Noobvious No obvious No obvious No obvious clinical clinical clinicalclinical observation observation observation observation

Clinical observations for dogs treated with compound 35 at 48 mg/kg areprovided in Table 86.

time D1501 D1502 D1502 point L-vehicle R-TA L-vehicle R-TA L-vehicleR-TA  8 hr No No obvious clinical No obvious No obvious No obvious Noobvious clinical obvious observation clinical clinical clinicalobservation clinical observation observation observation observation  24hr Swelling Red spots at groin Swelling No obvious Slight Slightswelling/red 4 * 4 cm 3 * 4 cm clinical swelling spot near theobservation saphenous vein of hind limb  48 hr Swelling Red spots atgroin Swelling No obvious Slight Slight swelling/red 4 * 4 cm 3 * 4 cmclinical swelling spot near the observation saphenous vein of hind limb 72 hr Swelling Swelling for the Swelling Swelling in the SwellingSwelling in the 4 * 4 cm whole leg; obvious 3 * 4 cm whole leg; 5 * 5 cmwhole leg; obvious swelling at upper leg obvious swelling at upper witharea of swelling at leg with area of 10 * 11 cm/Red spots inner upperleg 8 * 12 cm/red at the at groin/BT: 39.1° C. with volume of innerupper 15 * 13 * 3 cm leg/BT: 39.3° C. and red/BT: 39.6° C.  96 hrSwelling Swelling for the Swelling Swelling in the Swelling Swelling inthe 4 * 4 cm whole leg; obvious 3 * 4 cm whole leg; 5 * 5 cm whole leg;obvious swelling at upper leg obvious swelling at upper with area ofswelling at leg with area of 10 * 11 cm/Red spots inner upper leg 8 * 12cm/ at groin/BT: 39.0° C. with volume of induration and red 15 * 13 * 3cm at the inner upper and leg/BT: 39.3° C. red/BT: 38.9° C. 120 hrSwelling Swelling for the Swelling Swelling in the Swelling Swelling inthe 4 * 4 cm whole leg; obvious 3 * 4 cm whole leg; 5 * 5 cm whole leg;obvious swelling at upper leg obvious swelling at upper with area ofswelling at leg with area of 10 * 11 cm/Red spots inner upper leg 8 * 12cm/ at groin/BT: 39.1° C. with volume of induration and red 15 * 13 * 3cm at the inner upper and leg/red spot at calf/ red/BT: 39.6° C. BT:39.4° C. 144 hr Swelling Obvious swelling at Swelling Induration inSwelling Swelling in the 4 * 4 cm upper leg with area 3 * 4 cm the wholeleg; 5 * 5 cm whole leg; obvious of obvious swelling at upper 10 * 11cm/induration swelling at leg with area of 5 * 4 cm at the back innerupper leg 8 * 12 cm/ of upper leg with with volume of induration and redredness/induration 15 * 13 * 3 cm at the inner upper 9 * 7 cm at innerside and leg/red spot at calf/ of upper leg with redness/BT: 38.9° C.BT: 39.0° C. redness/Red spots at groin/BT: 38.7° C. 168 hr SwellingObvious swelling at Swelling Induration in Swelling Swelling in the 4 *4 cm upper leg with area 3 * 4 cm the whole leg; 5 * 5 cm whole leg;obvious of obvious swelling at upper 10 * 11 cm/induration swelling atleg with area of 5 * 4 cm at the back inner upper leg 8 * 12 cm/ ofupper leg with with volume of induration at the redness/induration 15 *13 * 3 cm back of upper leg 9 * 7 cm at inner side and with of upper legwith redness/BT: 39.9° C. redness/induration redness/Red spots at 8 * 6cm at the inner groin/BT: 38.9° C. upper leg/red spot at calf/BT: 39.3°C. 192 hr Swelling Obvious swelling at Swelling Induration in SwellingObvious swelling 4 * 4 cm upper leg with area 3 * 4 cm the whole leg;5 * 5 cm/red at upper leg with of obvious spots area of 8 * 12 cm/ 10 *11 cm/induration induration at induration at the 5 * 4 cm at the backinner upper leg back of upper leg of upper leg with with 15 * 13 cm/with redness/induration BT: 38.7° C. redness/induration 9 * 7 cm atinner side 8 * 6 cm at the inner of upper leg with upper leg/red spotredness/Red spots at at calf/BT: 39.2° C. groin/BT: 38.2° C. 216 hrSwelling Obvious swelling at Swelling Obvious Swelling induration at the4 * 4 cm upper leg with area 3 * 4 cm induration at 4 * 4 cm/red backand inner of inner upper leg spots uppper leg with 10 * 11 cm/indurationwith 13 * 9 cm/ 7 * 12 cm/red spot at 5 * 4 cm at the back BT: 39.2° C.calf/BT: 39.2° C. of upper leg with redness/induration 9 * 7 cm at innerside of upper leg with redness/Red spots at groin/BT: 38.6° C. 240 hrSwelling induration 5 * 4 cm at Swelling Obvious Swelling induration atthe shrinking the back of upper 3 * 4 cm induration at 4 * 4 cm/red backand inner leg with inner upper leg spots uppper leg withredness/induration with 13 * 9 cm/ 7 * 12 cm/red spot at 9 * 7 cm atinner side BT: 38.6° C. calf/BT: 38.9° C. of upper leg with redness/Redspots at groin/BT: 38.2° C. 264 hr Swelling induration3 * 4 cm atSwelling Obvious Swelling induration at the shrinking the back of upper3 * 3 cm induration at 2 * 2 cm/red back and inner leg with inner upperleg spots uppper leg with redness/induration6 * with 10 * 9 cm/ 6 * 6cm/BT: 38.7° C. 5 cm at inner side BT: 38.4° C. of upper leg withredness/Red spots at groin/BT: 38.0° C. 288 hr No induration3 * 4 cm atSwelling Obvious red spots induration at the obvious the back of upper3 * 3 cm induration at back and inner clinical leg with inner upper leguppper leg with observation redness/induration6 * with 9 * 9 cm/ 6 * 6cm/BT: 38.7° C. 5 cm at inner side BT: 38.6° C. of upper leg withredness/Red spots at groin/BT: 38.4° C. day 13 No induration3 * 4 cm atNo obvious Obvious No obvious induration at the obvious the back ofupper clinical induration at clinical back and inner clinical leg withobservation inner upper leg observation uppper leg with observationredness/Red spots at with 7 * 7 cm 6 * 6 cm groin day 14 Noinduration3 * 4 cm at No obvious Obvious No obvious induration at theobvious the back of upper clinical induration at clinical back and innerclinical leg with observation inner upper leg observation uppper legwith observation redness/Red spots at with 7 * 6 cm 6 * 6 cm groin day15 No induration3 * 4 cm at No obvious Obvious No obvious induration atthe obvious the back of upper clinical induration at clinical back andinner clinical leg with observation inner upper leg observation uppperleg with observation redness/Red spots at with 7 * 7 cm 6 * 6 cm groinday 16 No induration3 * 4 cm at No obvious Obvious No obvious indurationat the obvious the back of upper clinical induration at clinical backand inner clinical leg with observation inner upper leg observationuppper leg with observation redness/Red spots at with 6 * 6 cm 6 * 6 cmgroin day 17 No induration3 * 2 cm at No obvious Obvious No obviousinduration at the obvious the back of upper clinical induration atclinical back and inner clinical leg with observation inner upper legobservation uppper leg observation redness/Red spots at with 6 * 6 cmwith4 * 6 cm groin day 18 No induration3 * 2 cm at No obvious Obvious Noobvious induration at the obvious the back of upper clinical indurationat clinical back and inner clinical leg with observation inner upper legobservation uppper leg observation redness/Red spots at with 5 * 5 cmwith4 * 3 cm groin day 19 No redness/Red spots at No obvious Obvious Noobvious induration at the obvious groin clinical induration at clinicalback and inner clinical observation inner upper leg observation uppperleg observation with 4 * 5 cm with2 * 3 cm day 20 No redness/Red spotsat No obvious Obvious No obvious Not obvious obvious groin clinicalinduration at clinical induration clinical observation inner upper legobservation observation with 4 * 4 cm

We claim:
 1. A compound, or pharmaceutically acceptable salt thereof,having a structure provided below:

wherein X is O or CH₂.
 2. A compound, or pharmaceutically acceptablesalt thereof, wherein the compound is(4aS,7aS,12bS)-34cyclopropylmethyl)-4a-hydroxy-7-methylene-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(E)-octadec-9-enoate.
 3. A compound, or pharmaceutically acceptable saltthereof, wherein the compound is(4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a-hydroxy-7-oxo-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-9-yl(E)-octadec-9-enoate.
 4. A pharmaceutical composition comprising acompound of claim 1, or pharmaceutically acceptable salt thereof, and atleast one pharmaceutically acceptable excipient.
 5. The pharmaceuticalcomposition of claim 4, wherein the pharmaceutically acceptableexcipient is cottonseed oil.
 6. The pharmaceutical composition of claim4, wherein the pharmaceutically acceptable excipient is sesame oil.
 7. Apharmaceutical composition comprising a compound of claim 2, orpharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable excipient.
 8. The pharmaceutical compositionof claim 7, wherein the pharmaceutically acceptable excipient iscottonseed oil.
 9. The pharmaceutical composition of claim 7, whereinthe pharmaceutically acceptable excipient is sesame oil.
 10. Apharmaceutical composition comprising a compound of claim 3, orpharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable excipient.
 11. The pharmaceuticalcomposition of claim 10, wherein the pharmaceutically acceptableexcipient is cottonseed oil.
 12. The pharmaceutical composition of claim10, wherein the pharmaceutically acceptable excipient is sesame oil.